Structure-based peptide inhibitors of alpha-synuclein aggregation

ABSTRACT

This invention relates to inhibitory peptides which bind to α-synuclein molecules and inhibit α-synuclein amyloidogenic aggregation, α-synuclein cytotoxicity, and spread of α-synuclein. Methods of making and using the inhibitory peptides (e.g. to treat subjects having conditions or diseases that are mediated by α-synuclein, such as Parkinson&#39;s disease, dementia with Lewy bodies, or MSA) are described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application that claims the benefitunder 35 U.S.C. § 121 of U.S. patent application Ser. No. 16/883,515,filed May 26, 2020, which claims the benefit under 35 U.S.C. § 121 ofU.S. patent application Ser. No. 16/311,593, filed Dec. 19, 2018, whichis the National Stage of International Application No. PCT/US2017/040106(International Publication No. WO 2018/005867), filed Jun. 29, 2017,which claims priority under Section 119(e) of co-pending U.S.Provisional Patent Application Ser. No. 62/356,410, filed Jun. 29, 2016,entitled “STRUCTURE-BASED PEPTIDE INHIBITORS OF ALPHA-SYNUCLEINAGGREGATION” the contents of which are incorporated herein by reference.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant NumberAG029430, awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 9, 2017, isnamed 30435_305-WO-U2_SL.txt and is 18,456 bytes in size.

BACKGROUND INFORMATION

Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiplesystem atrophy (MSA) are together classified as Synucleinopathies, aclass of neurodegenerative diseases characterized by the pathologicalaccumulation of the protein, α-synuclein (α-syn) in neuronal cells.Together these make up the second most common form of neurodegenerativedisease. The presynaptic protein α-synuclein (α-syn), found in bothsoluble and membrane-associated fractions of the brain, aggregates in,for example, Parkinson's Disease (PD). These aggregates are the maincomponent of Lewy bodies, the defining histological feature of thisneurodegenerative disease, and have been shown to accompany neuronaldamage⁷. Without wishing to be bound by any particular mechanism, it issuggested that this and two other observations point to aggregated α-synas a molecular cause of Parkinson's disease⁸. The first is that familieswith inherited forms of PD carry mutations in α-syn, such as A53T, andabundant Lewy bodies ^(9,10,11) The second is that families withduplicated or triplicated genes encoding α-syn develop early onset PD,presumably because at high local concentrations α-syn is forced intoamyloid ^(12, 13).

Based on structural studies, a number of different models for α-synfibrils have been proposed. Limited proteolysis and NMR studies suggestthat the fibril core is composed of residues 30-100 (Miake H (2002) JBiol Chem 277(21):19213-19219). Crystal structures and NMR studiessuggest different models of α-syn fibrils. In one model based on crystalstructures of short segments, two monomers per sheet form extendedsteric zippers (Rodriguez J A, et al. (2015) Nature 525(7570):486-490).In a second ssNMR-based model, a greek key topology with one monomer peramyloid layer has been shown (Tuttle Md., et al. (2016) Nat Struct MolBiol 23(5):409-415). Taken together these studies suggest that α-syn canform polymorphic fibrillar architectures. The segment 68-78 of α-syntermed NACore may form the core of α-syn fibrils. NACore, resides withinthe 35-residue NAC (non-amyloid β component) domain found in amyloiddeposits (Rodriguez J A, et al. (2015) Nature 525(7570):486-490). NACoreaggregates readily, and the aggregates display properties such asdiffraction pattern and cytotoxicity similar to full-length α-syn.Additionally, β-synuclein, a homologue does not contain residues 74-84and is not found in amyloid deposits, and removal of residues 71-82 hasbeen shown previously to reduce the aggregation and toxicity in vitroand in a drosophila model (Giasson et al. (2001) J Biol Chem276(4):2380-2386 and Periquet et al. (2007) J Neurosci27(12):3338-3346). Additionally, a modification at Thr72 prevents itsaggregation propensity.

The present inventors recently showed that it is possible to efficientlyarrest the aggregation of the Alzheimer's Disease related protein Tauand the semen-derived enhancer of HIV virus infection (SEVI) utilizingshort amino-acid inhibitors designed to specifically “cap” the growingaggregates (Sievers et al.³⁶; U.S. Pat. No. 8,754,034⁵⁷). They alsoshowed that the same design strategy can lead to a designedcell-penetrating peptide that inhibits p53 amyloid formation, rescuesmutant p53 function in cancer cell lines and decreases tumorproliferation (US patent application 2014/037387⁵⁸). Accordingly, thereis an attractive therapeutic window which targets the α-synucleinamyloid aggregation, major component of intracellular deposits in theform of Lewy bodies (LBs) in Parkinson's disease and relatedneurodegenerative disorders.

There is a need to identify agents which prevent and/or inhibitα-synuclein aggregation and/or cytotoxicity.

SUMMARY OF THE INVENTION

Although α-syn amyloid formation has been extensively characterized,little headway has been made in developing therapeutics that can inhibitα-syn aggregation or reduce the prion-like spread of α-syn aggregates(“seeds”) from cell to cell. Promising approaches include antibodiesthat sequester α-syn aggregates as well as small molecule stabilizersthat bind α-syn monomers (see, e.g. Mandler M, et al. (2015) MolNeurodegener 10(1). doi:10.1186/s13024-015-0008-9; Wrasidlo W, et al.(2016) Brain:aww238). Using the atomic structure of NACore as atemplate, we have developed a new class of inhibitors, peptide agentsthat bind α-syn seeds and prevent their growth and elongation. As shownbelow, these inhibitors inhibit α-synuclein fibril formation and seedingin a number of model systems, and spread of a-synuclein aggregates.

The invention disclosed herein has a number of embodiments. Oneembodiment of the invention is a composition of matter comprising atleast one inhibitory peptide that inhibits α-synuclein (SEQ ID NO: 1)aggregation by binding to residues 68-78 of α-synuclein. In typicalembodiments of the invention, the inhibitory peptide comprises thesequence GAVVWGVTAVKK (SEQ ID NO: 3) or RAVVTGVTAVAE (SEQ ID NO: 4).Optionally the inhibitory peptide comprises the sequence GAVVWGVTAVKKKKK(SEQ ID NO: 5), GAVVWGVTAVKKGRKKRRQRRRPQ (SEQ ID NO: 6); orYGRKKRRQRRRAVVTGVTAVAE (SEQ ID NO: 7). In certain embodiments of theinvention, the composition comprises a plurality of inhibitory peptides.Typically, the inhibitory peptide(s) is/are from 6 to 30 amino acids inlength.

In the inhibitory peptide compositions of the invention, at least one ofthe amino acids in the inhibitory peptide may comprise a non-naturallyoccurring amino acid (e.g. a D-amino acid or an amino acid comprising aN-methyl group moiety); and/or the inhibitory peptide is coupled to aheterologous peptide tag. Such heterologous peptide tags include aminoacid sequences that increase peptide solubility; or amino acid sequencesthat facilitate monitoring or manipulation of the peptide; or amino acidsequences that facilitate peptide entry into a mammalian cell.Optionally these peptide compositions include a pharmaceuticallyacceptable carrier and a peptide stabilizing excipient.

Another embodiment of the invention is an expression vector encoding aninhibitory peptide that inhibits α-synuclein aggregation by binding toresidues 68-78 of α-synuclein. A related embodiment is a kit comprisinga peptide inhibits α-synuclein (SEQ ID NO: 1) aggregation by binding toresidues 68-78 of α-synuclein or an expression vector encoding such apeptide. Embodiments of the invention also include a method of making apeptide disclosed herein by synthesizing it chemically or producing itrecombinantly. Yet another embodiment of the invention is a complexcomprising α-synuclein and a peptide that inhibits α-synucleinaggregation by binding to residues 68-78 of α-synuclein.

Yet another embodiment of the invention is a method for reducing orinhibiting α-synuclein (SEQ ID NO: 1) aggregation, comprising contactingα-synuclein amyloid fibrils with an inhibitory peptide disclosed hereinin an amount sufficient to reduce or inhibit α-synuclein aggregation.Optionally in this method, the α-synuclein amyloid fibrils are within anin vivo environment. Alternatively in this method, the α-synucleinamyloid fibrils are within an in vitro environment. A related embodimentof the invention is a method of modulating the size or rate of growth ofa α-synuclein amyloid fibril, comprising contacting the fibril with anamount of at least one inhibitory peptide that inhibits α-synuclein (SEQID NO: 1) aggregation by binding to residues 68-78 of α-synuclein in anenvironment where the inhibitory peptide contacts residues 68-78 ofα-synuclein so that the contacted α-synuclein amyloid fibril exhibits amodulated size or rate of growth.

Yet another embodiment of the invention is a method of observing thepresence or absence of α-synuclein amyloid fibrils in a biologicalsample comprising combining a biological sample with a peptide thatbinds to residues 68-78 of α-synuclein, allowing the peptide to bind toα-synuclein amyloid fibrils that may be present in the biologicalsample, and then monitoring this combination for the presence ofcomplexes formed between α-synuclein amyloid fibrils and the peptide;wherein the presence of said complexes show the presence of α-synucleinamyloid fibrils in the biological sample. In this embodiment, one of ourinhibitors can be bound to an imaging agent such as a radioactive label,a radio-opaque label, a fluorescent dye, a fluorescent protein, acolorimetric label or the like (e.g. to facilitate an imaging methodsuch as MRI or PET), and our inhibitor will bind to alpha-synucleinfibrils in the brain of a patient, permitting imaging of the fibrils fordiagnosis for following disease progression.

Other objects, features and advantages of the present invention willbecome apparent to those skilled in the art from the following detaileddescription. It is to be understood, however, that the detaileddescription and specific examples, while indicating some embodiments ofthe present invention, are given by way of illustration and notlimitation. Many changes and modifications within the scope of thepresent invention may be made without departing from the spirit thereof,and the invention includes all such modifications.

DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor.

FIG. 1 : Design of α-syn seeding inhibitors. (A) Structure based designof α-syn aggregation inhibitors. Structure of NACore is composed of twoself complementary β-sheets forming steric zipper. Three types ofinhibitors (maroon, orange and cyan) were identified that bind one orboth ends. (B) Binding energies of the different inhibitors calculatedby Rossetta show S37 and S62 bind both interfaces while S71 is onlypredicted to bind one interface. Shape complementarity of all threeinhibitors is high. (C)-(E) graphed data from Thioflavin T assaysmeasuring α-syn aggregation and the effect of inhibitors. 50 μM α-synand inhibitors at 5 fold molar excess were added.

FIG. 2 : α-syn aggregates formed in the presence of inhibitors are notseeding competent. (A) Experimental design of cell culture seedingassay. α-syn was aggregated in the presence of inhibitors and themixture was transfected in HEK293 cells expressing YFP labeled WT α-synor A53T α-syn (green). A53T is a familial variant of alpha-synucleinwhich causes early onset Parkinson's disease. Upon transfectionendogenous α-syn formed fluorescent puncta (red) (B) NACore does notaffect seeding capacity of α-syn fibrils. (C,D,E) 50 μM α-syn aggregatedin 10, 5, 2 and 1 fold excess of S37, S61 and S62 was not seedingcompetent as measured by counting total number of particles formed perwell in both WT and A53T expressing cells. (F) 50 μM α-syn aggregated inexcess of S71 was not seeding competent as measured by counting totalnumber of particles formed per well in both WT and A53T expressingcells. Results shown as Mean±SD (n=3). Statistical significance wasanalyzed by two way ANOVA.

FIG. 3 : Inhibitors prevent seeding in cell culture. (A) Experimentaldesign of cell culture seeding assay. 125 nM recombinant α-syn fibrilswere transfected with different amounts of inhibitors and aggregationmonitored over time. (B) NACore does not affect seeding capacity (C, D,E) S37, S61, S62 and S71 reduce seeding capacity of α-syn. All datareported as particles counted per well and normalized to particlescounted in buffer treated wells. Results shown as Mean±SD (n=3).Statistical significance was analyzed by two way ANOVA.

FIG. 4 : Extracted filaments from PD brain tissue seed α-syn aggregationin vitro and in cell culture. (A) Protocol for extraction of sarkosylinsoluble protein filaments from PD brain tissues. (B,C,D) 2% seeds from3 different subjects induce rapid α-syn aggregation with 4-10 foldincrease in ThT fluorescence. (E,F,G) α-syn seeded by filaments from PDsubjects induce more particles than α-syn alone. All data reported asparticles counted per well and normalized to particles counted in buffertreated wells. Results shown as Mean±SD (n=3). Statistical significancewas analyzed by two way ANOVA.

FIG. 5 : Inhibitors prevent seeding by PD tissue-extracted filaments.Filaments from 4 different subjects were tested for seeding α-synaggregation and monitored by ThT assay. S61 and S62 were effectiveagainst all seeds that were tested. S71 was effective against seeds Band D.

FIG. 6 : α-syn fibrils formed in the presence of PD filament seeds andinhibitors are not seeding competent. (A) Experimental design of cellculture seeding assay (B, C, D) Inhibitors were tested for inhibition ofseeding by three different PD brain extracted tissues. S71 was effectiveagainst all three seeds while S61 was effective against Seeds B. Alldata reported as particles counted in each well normalized to theparticles counted in buffer treated wells. Results shown as Mean±SD(n=3). Statistical significance was analyzed by two way ANOVA.

FIG. 7 : Inhibitors reduce seeding by PD filament seeded α-syn fibrilsin cell culture. (A) Experimental design of cell culture seeding assay.(B,C,D) α-syn fibrils formed in the presence of two different PD relatedfilaments were transfected in YFP-α-syn HEK cells and fluorescencemeasured thereafter for up to 6 days. S37 prevented seeding atconcentrations of 12.5 μM-1.25 μM. S61 prevented seeding with apronounced effect on Day 2. S71 reduced seeding at concentrations of6.25 μM. All data reported as number of particles counted per wellnormalized to particles counted on Day 0 before transfection. Resultsshown as Mean±SD (n=3). Statistical significance was analyzed by two wayANOVA.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. Many of the techniques and procedures describedor referenced herein are well understood and commonly employed usingconventional methodology by those skilled in the art. All publications,patents, and patent applications cited herein are hereby incorporated byreference in their entirety for all purposes. In the description of thepreferred embodiment, reference may be made to the accompanying drawingswhich form a part hereof, and in which is shown by way of illustration aspecific embodiment in which the invention may be practiced. It is to beunderstood that other embodiments may be utilized and structural changesmay be made without departing from the scope of the present invention.

α-synuclein, a 140 amino acid protein is found in amyloid deposits inneuronal cells in disease conditions. A causative link between α-synamyloid formation and disease progression is supported by the findingsthat gene duplications and familial mutations that increase amyloid loadalso cause early-onset PD, and more than 90% sporadic PD patients stainpositive for α-syn deposits. The invention disclosed herein provides astructure-based approach to halt α-syn aggregation. We hypothesized thatthe atomic structures of NACore and PreNAC are preserved in α-synamyloid seeds and recruit the endogenous protein into a zipperconformation. Using the atomic structure of NACore, we developedinhibitors that hinder fibril formation and tested their efficacy invitro and in cell culture. The same procedure can be used to developinhibitors based on PreNAC. The inhibitors are optimized to cap the endsof the fibril, preventing further addition of monomers. We used thesoftware Rosetta to design peptide sequences that interact favorablywith the NACore segment. The energy function used by Rosetta takes intoaccount electrostatic interactions, hydrogen bonding, Van der Waal sforces, among other terms to assess binding energy. Once a specificresidue has been shown to produce favorable binding in a certainposition of the designed peptide, it can be fixed and prevented fromfurther design, while the rest of the sequence is further refined. Weperformed this process of fixing and redesign iteratively until anoptimal set of sequences was identified. This rational design processallowed for computationally sampling of orders of magnitude moreinhibitor sequences than what was experimentally feasible to test.

We carried out screens for crystals of peptide segments within the NACdomain and adjacent regions, seeking structural information on themolecular basis of aggregation and toxicity of α-syn. Using the ZipperDBalgorithm ⁵⁵, crystallizable amyloid-forming segments in the NAC domainwere identified. In particular, we identified and concentrated on acentral segment of α-syn, residues 68-78, which is referred to herein asNACore because of its important role in both the aggregation andcytotoxicity of α-syn, NACore is the fibril-forming core of the 35residue NAC (Non Amyloid-β Component) domain (residues 61-95) of full,140 residue α-synuclein. In some studies, we utilized a sub-fragment ofNACore, comprised of residues 69-77. In other studies we crystallizedand determined the structure of residues 47-56 of α-synuclein termedPreNAC which forms another steric zipper. The identified segments werechemically synthesized and crystallized and their three-dimensionalstructures were determined by micro-crystallography and micro-electrondiffraction (MicroED).

To find the most effective inhibitors, nearly 100 different designs weretested empirically with sequential rounds of optimization on theinhibitor design. For example, we observed that the location of the tagon N or C terminus can affect its efficiency. Additionally, the type ofmodification added can also affect its efficacy. In our case, only a Trpsubstitution at Thr72 was effective whereas Arg substitution was not.Although the computational approach is not powerful enough to identifyone successful design, it can narrow our search for candidateinhibitors, which can then be refined through rational design.

The efficiency of capping inhibitors in preventing seeding was testedusing a cell-based assay. In this system, transfection of nanomolaramounts of α-syn seeds caused endogenous protein aggregation. Theaggregates display amyloidogenic properties—binding to amyloid-specificsmall molecules, faithfully transferred upon cell division andremarkable specificity (Sanders D W, et al. (2014) Neuron82(6):1271-1288). For example, α-syn fibrils can only seed α-syn proteininto aggregates. Notably, in this system we do not observe acute celldeath upon formation of aggregates with only a mild slowing of cellproliferation. Our inhibitors prevented puncta formation in this systemwith a single administration of inhibitors effective for 2-3 days.

Seeding, in the context of amyloid disease, is the sequential transferof pathologic protein aggregates along connected tissues. This processcontributes to progression and severity of neurodegenerative diseases.To date, there are no therapeutics that specifically target seeding, inpart, due to lack of information of the structural properties ofpathological seeds. The present application relates, e.g., to thedesign, synthesis and functional characterization of peptides that bindspecifically to α-synuclein (α-syn) aggregates and block, inhibit and/ordiminish α-syn aggregation and/or α-syn cytotoxicity. The peptideinhibitors specifically “cap” the growing aggregates of α-synuclein. Insome embodiments, the peptides are fused to cell penetrating peptidesthat enhance their delivery into cells.

Apart from the spontaneous aggregation of intracellular α-syn intoamyloid fibrils, a second phenomenon that contributes to diseaseprogression is the prion-like spread of α-syn aggregates (Goedert M(2015). Science 349(6248):1255555-1-1255555-9). Braak staging has shownthat pathology gradually spreads over time through connected brainregions, and cell culture and animal models show that small amounts ofα-syn aggregates can act as seeds and induce aggregation of the nativeprotein (see, e.g. Braak H, et al. (2003) Neurobiol Aging 24(2):197-211;Braak et al. (2009) Adv Anat Embryol Cell Biol 201:1-119;Masuda-Suzukake M, et al. (2013) Brain 136(4):1128-1138; Desplats P, etal. (2009) Proc Natl Acad Sci USA 106(31):13010-13015; Luk K C, et al.(2009) Proc Natl Acad Sci 106(47):20051-20056). Although distinct fromcanonical prions that can be transmitted from person to person, thisphenomenon of ‘seeding’ seems the driver of disease progression.

Patient-extracted fibrils are observed to differ in seeding capacity anddisplay strain-like characteristics. In vitro the PD patient extractedfibrils caused dramatic increase in α-syn aggregation, and in cellculture model the seeded samples increased puncta formation. Notably,unlike previous reports where patient derived α-syn filaments seeds incell culture, in our assays we did not observe seeding in cell culture(Prusiner S B, et al. (2015) Proc Natl Acad Sci 112(38):E5308-E5317; andWoerman A L, et al. (2015) Proc Natl Acad Sci 112(35):E4949-E4958).Previous reports utilized substantia nigra tissues whereas we usedfrontal and temporal tissues, which might differ in seeding potency.Indeed in previous reports, fibrils extracted from different brainregions have been shown to differ in seeding capacity reminiscent ofdifferent strains (Prusiner S B, et al. (2015) Proc Natl Acad Sci112(38):E5308-E5317). Furthermore, the different inhibitors varied inefficiency against different seeds. For example, S61 was effectiveagainst seeds A and B only whereas S71 was effective against seeds B andD. Recently, an NMR structure of full length α-syn fibrils was reportedin which the NACore segment was not found in an extended zipperconformation although the segment 68-78 is found in the core of thefibril (Tuttle Md., et al. (2016) Nat Struct Mol Biol 23(5):409-415). Orit may be that the inhibitors described therein can bind to theconformation of NACore in the NMR structure. Also it is conceivable thatthe NMR structure and the steric zipper structure are differentpolymorphs. In the absence of a diagnostic method to identify differentpolymorphs in human subjects, theoretically a cocktail of differentinhibitors targeting different polymorphs could be useful.

We used a combination of computational methods and rational design todevelop a line of inhibitors targeted at preventing the spread of α-synaggregates. Our approach was only made possible by the determination ofthe atomic structure of the core of α-syn amyloid fibrils, and thisapproach can be adopted for other diseases where seeding plays a role indisease progression. The inhibitors prevent aggregation of α-synucleinin vitro and in cell culture models. The inhibitors also show efficacyin preventing seeding by patient-derived α-synuclein fibrils both invitro and in cell culture models. Our results provide evidence thatpathological seeds of α-syn contain steric zippers and suggest atherapeutic approach targeted at the spread and progression that may beapplicable for PD and related synucleinopathies. Similarly ourinhibitors may be applicable for a diagnostic approach for PD andrelated synucleinopathies.

We hypothesized that mutations, overexpression or other cellular factorscan destabilize the native α-synuclein structure, exposing an adhesive,“steric-zipper” segment, proposed as the basic building block of amyloidaggregates ^(18, 19). We therefore generated high-resolution views ofthe amyloid spines of α-synuclein aggregates. We then applied theRosetta-based method ³⁶ to design inhibitors which specifically “cap”the growing aggregates of α-synuclein and thus disrupt and inhibitfurther α-synuclein aggregation, using the α-synuclein 69-77 structureor the α-synuclein 68-78 structure as a template.

We utilized the atomic structure of NACore [68-GAVVTGVTAVA-78] (SEQ IDNO: 46) as a template and using computational and structure-basedapproaches designed peptidic inhibitors. The atomic structure of NACorerevealed a pair of self-complementary β-sheets forming a steric zipper(Sawaya M R, et al. (2007) Nature 447(7143):453-457). The inhibitors arepredicted by Rossetta-based computational modeling to bind the stericzipper interface and ‘cap’ the fibrils. We identified 3 candidateinhibitors; S37, S61 and S71 that bind favorably with one or both endsof the zipper (FIG. 1 ). The binding energies and shape complementarityof the three inhibitors are also favorable (FIG. 1B). All the inhibitorsretain most residues of the native sequence of NACore but contain one ormore modified residues. Rodriquez et al. showed that a smaller 9-residuesegment within NACore [69-AVVTGVTAV-77] (SEQ ID NO: 48) aggregatesslower than NACore and the structure is similar to NACore. Rodriguez etal. (2015) also describes a second segment of α-syn, termed PreNAC, withsequence GVVHGVTTV residues 47-56. Designed inhibitors to this sequencemight also inhibit fibril formation of α-syn.

In order to prevent the self-aggregation of our designed inhibitors, weused the shorter segment along with one or more modifications. S37 has aW mutation at Thr72 and an additional poly-lysine tag at the C-terminusto induce charge-charge repulsion. It is predicted to bind both tips ofthe steric zipper fibril. S61 and S62 retain the same inhibitor sequenceas S37 but instead of poly-lysine tag, a TAT tag is added to aidsolubility and prevent self-aggregation. S71 has a methylated glycine atGly73 that weakens hydrogen bonding along the β-sheet and an additionalTAT tag for solubility and cell penetration.

We tested the efficacy of the inhibitors in an in vitro aggregationassay. Recombinantly purified α-syn was aggregated in the presence ofthe inhibitors and monitored by measuring fluorescence of Thioflavin T,an amyloid binding dye. All three inhibitors prevented aggregation witha significant reduction in ThT fluorescence (FIG. 1C, 1D, 1E).

We tested the efficacy of the inhibitors in preventing aggregation in acell culture model. For these assays, we utilized two HEK293T cells thatstably express YFP-labeled full-length WT α-syn and A53T α-syn (SandersD W, et al. (2014) Neuron 82(6):1271-1288). In this model,lipofectamine-mediated transfection of recombinant fibrils leads toaggregation of the endogenous YFP-labeled protein that are seen asfluorescent puncta. Additionally, these puncta increase in size andnumber over time. This proliferation of aggregates over time isindicative of a ‘seeding’ phenomenon whereby a small amount of amyloidfibrils induces aggregation of the endogenous protein. First we testedthe parent peptide segment, NACore to check its effect on seeding. α-synwas aggregated in the presence of molar excess of NACore (FIG. 2B). Themixture was transfected in cells and puncta formation was visualized andthe number of puncta were counted as particles per well. As expected,NACore did not cause a significant reduction in puncta formation ineither cell line. Next we aggregated 50 μM α-syn in the presence of 500μM, 250 μM, 100 μM and 50 μM inhibitor corresponding to 10, 5, 2 and 1fold excess. The mixture was then transfected in cells and aggregationwas monitored over time for up to 3 days by fluorescence imaging (FIG.2A). S37 caused a significant reduction in seeding for up to 2 days inboth WT and A53T expressing cell lines. Similar to S37, S61 (FIG. 2D)also caused a reduction in puncta formation with maximum efficacy at 2,5 and 10 fold excess in both cell lines. Aggregates formed in thepresence of S62 (FIG. 2E) were also seeding incompetent withsignificantly less particles forming at all inhibitor concentrations.S71 was tested at equimolar and sub-stoichiometric ratios and found toreduce the seeding potency of the aggregates (FIG. 2F). These resultssuggest that the inhibitors prevent formation of seeding competentaggregates.

We tested the efficacy of the inhibitors to prevent seeding in the cellculture model (FIG. 3A). We transfected the α-syn fibrils along with thedifferent inhibitors. NACore, the parent aggregating peptide did notaffect the seeding potency of α-syn fibrils (FIG. 3B). S37 causedsignificant reduction in seeding in both cell lines at 12.5 11M and 6.25μM concentrations for up to 2 days (FIG. 3C). S61 at concentrations of2.5 11M and 1.25 μM caused a prolonged reduction of seeding lasting upto 6 days in WT α-syn cells and 2 days in A53T α-syn HEK cells (FIG.3D). S62 was effective at concentrations of 12.5 μM and 6.25 μM in bothcell lines (FIG. 3E). S71 was effective at low concentrations of 1.25Interestingly, we observed that addition of higher concentrations ofthese inhibitors does not prevent seeding suggesting a criticalconcentration range with maximum efficacy. Together these resultssuggest that inhibitors can cap fibril seeds and prevent theirelongation.

We extracted insoluble protein aggregates from frozen autopsy PD braintissues. We obtained tissues from 4 different subjects including thesubstantia nigra and frontal regions of one subject and temporal andfrontal regions of other subjects. Using previously described protocols(Goedert et al. (1992) Neuron 8(1):159-168) that included precipitationwith the ionic detergent sarkosyl, we extracted insoluble proteinaggregates (FIG. 4A). All samples robustly seeded α-syn aggregation invitro and in our cell culture model. In vitro addition of 2% seedsincreased the ThT fluorescence 4 to 10 fold (FIG. 4B, 4C, 4D) along witha small decrease in the lag time. The seeded samples were thentransfected in HEK cells. Consistent with the ThT assay, all seededsamples induced rapid puncta formation (FIG. 4E, 4F, 4G). Thus, fibrilsextracted from PD brain tissues seeds recombinant protein, and theaggregates formed upon seeding induce rapid puncta formation in cellculture.

We tested the effect of the different inhibitors in preventing α-synaggregation in the presence of PD extracted seeds. S71 and S62 were mosteffective showing efficacy against all seeds as measured by ThTfluorescence assay (FIG. 5 ). S61 also reduced aggregation of twodifferent seeds (FIG. 5B, 5D) whereas S37 showed marginal reduction inThT fluorescence (FIG. 5D). Next we tested the seeding potency of theα-syn aggregates formed in the presence of PD seeds and the differentinhibitors (FIG. 6A). Aggregates formed in the presence of S71 (FIGS.6B, 6C and 6D) did not induce puncta formation in both WT and A53Texpressing HEK cells. S61 also showed efficacy (FIG. 6C). Consistentwith the in vitro assay, S37 was not effective in reducing the seedingpotency of aggregates. Together these results suggest that S62 and S71can prevent formation of seeding competent fibrils.

We tested the efficacy of the inhibitors in preventing seeding by PDfibrils in cell culture. α-syn aggregates formed in the presence of PDfibrils were transfected in WT HEK cells along with the differentinhibitors (FIG. 7A). S37 prevented puncta formation for up to 2 daysfor two different PD filaments at concentrations of 12.5 μM-1.25 μM(FIG. 7B). Similar to S37, S61 also showed efficacy for up to 2 days atconcentrations of 12.5 μM-1.25 μM, and S71 also prevented seeding atsimilar concentrations. These results suggest that inhibitors robustlyprevent seeding in cell culture.

The disclosure immediately above describes work with a number of workingembodiments of the invention. The invention disclosed herein providessuch inhibitory peptides; pharmaceutical compositions comprisinginhibitory peptides of the invention and a pharmaceutically acceptablecarrier; methods of using the inhibitory peptides to block, inhibitand/or prevent α-synuclein aggregation and/or α-synuclein cytotoxicity,comprising contacting an α-synuclein molecule (e.g. a monomer, smallaggregate, oligomer, or fibril) with an effective amount of a peptideinhibitor of the invention, or administering to a subject an effectiveamount of a peptide inhibitor of the invention; and computer-relatedembodiments, such as a method for designing and obtaining inhibitorypeptides or small molecules based on the structures described herein.

Advantages of the inhibitory peptides of the invention include: (1)Synthetic peptides are not expensive. (2) Cell penetration and proteinstability are not challenging thanks to their composition and smallsize. In addition, the peptides can be fused to cell penetratingpeptides that enhance their delivery into cells. (3) They areunexpectedly stable: they are not proteolyzed and exhibit a sufficientlylong half-life to function in vivo (e.g. in a body). (4) Peptideinhibitors are specific for their targets, and therefore present feweropportunities for side effects than, e.g., small molecules, which maybind to many targets.

The invention disclosed herein has a number of embodiments. Oneembodiment of the invention is a composition of matter comprising atleast one inhibitory peptide that inhibits α-synuclein (SEQ ID NO: 1)aggregation by binding to residues 68-78 of α-synuclein. As disclosed inthe Examples below, working embodiments of these peptides include S37,S62 and S71 as shown in Table 1 below:

Inhibitor Sequence S37 GAVVWGVTAVKKKKK S62 GAVVWGVTAVKKGRKKRRQRRRPQ S71YGRKKRRQRRRAVVT{N-me-Gly}VTAVAE

In the table N-me-Gly stands for Glycine with a methylated amino group.Bold type gives the inhibitor sequence. Unbold type shows tags, eithersolubilizing tags or linkers.

In typical embodiments of the invention, the inhibitory peptidecomprises the sequence GAVVWGVTAVKK (SEQ ID NO: 3) or RAVVTGVTAVAE (SEQID NO: 4). Optionally the inhibitory peptide comprises the sequenceGAVVWGVTAVKKKKK (SEQ ID NO: ⁵), GAVVWGVTAVKKGRKKRRQRRRPQ (SEQ ID NO: 6);or

YGRKKRRQRRRAVVTGVTAVAE (SEQ ID NO: 7). In certain embodiments of theinvention, the composition comprises a plurality of inhibitory peptides.Typically, the inhibitory peptide(s) is/are from 6 to 30 amino acids inlength. Active variants of any of these inhibitory peptides are alsoincluded. Inhibitory peptides having the preceding sequences, includingthe active variants, are sometimes referred to herein as “inhibitorypeptides of the invention.”

In the inhibitory peptide compositions of the invention, at least one ofthe amino acids in the inhibitory peptide comprises a non-naturallyoccurring amino acid (e.g. a D-amino acid or an amino acid comprising aN-methyl group moiety); and/or the inhibitory peptide is coupled to aheterologous peptide tag. Such heterologous peptide tags include aminoacid sequences that increase peptide solubility in vivo or in vitro(e.g. a plurality of arginine residues); or amino acid sequences thatfacilitate monitoring or manipulation of the peptide in vivo or in vitro(a plurality of lysine or histidine amino acids); or amino acidsequences that facilitate peptide entry into a mammalian cell (e.g. acell penetrating peptide sequence). Another aspect of the invention is apharmaceutical composition comprising an inhibitory peptide of theinvention and a pharmaceutically acceptable carrier. Such pharmaceuticalcompositions are sometimes referred to herein as “pharmaceuticalcompositions of the invention.” Optionally the peptide compositionsdisclosed herein include a pharmaceutically acceptable carrier and apeptide stabilizing excipient.

Another embodiment of the invention is an expression vector encoding aninhibitory peptide that inhibits α-synuclein aggregation by binding toresidues 68-78 of α-synuclein. Optionally, the expression vector is oneused to deliver polypeptides to mammalian cells such as a lentivirus. Inthis context, another embodiment of the invention is a method ofdelivering a DNA encoding an inhibitory peptide that inhibitsα-synuclein aggregation in a mammalian cell (e.g. an ex vivo or in vivocell) by contact the mammalian cell with a vector that transduces thecell so that he DNA is expressed in the cell.

Another embodiment is kit comprising a peptide inhibits α-synuclein (SEQID NO: 1) aggregation by binding to residues 68-78 of α-synuclein or anexpression vector encoding such a peptide. Embodiments of the inventionalso include a method of making a peptide disclosed herein bysynthesizing it chemically or producing it recombinantly. Yet anotherembodiment of the invention is a complex comprising α-synuclein and apeptide that inhibits α-synuclein aggregation by binding to residues68-78 of α-synuclein.

Another embodiment is a peptide designed on the structure of PreNAC(residues 47-56 of α-synuclein: GVVHGVTTVA) to inhibit fibril formationof α-synuclein.

Yet another embodiment of the invention is a method for reducing orinhibiting α-synuclein (SEQ ID NO: 1) aggregation, comprising contactingα-synuclein amyloid fibrils with an inhibitory peptide disclosed hereinin an amount sufficient to reduce or inhibit α-synuclein aggregation.Optionally in this method, the α-synuclein amyloid fibrils are within anin vivo environment. Alternatively in this method, the α-synucleinamyloid fibrils are within an in vitro environment. A related embodimentof the invention is a method of modulating the size or rate of growth ofa α-synuclein amyloid fibril, comprising contacting the fibril with anamount of at least one inhibitory peptide that inhibits α-synuclein (SEQID NO: 1) aggregation by binding to residues 68-78 of α-synuclein in anenvironment where the inhibitory peptide contacts residues 68-78 ofα-synuclein so that the contacted α-synuclein amyloid fibril exhibits amodulated size or rate of growth.

The peptide inhibitors disclosed herein can be used as a therapy to haltthe spread of Parkinson's disease within the brain. Alternatively, thepeptide inhibitors disclosed herein can be used as a diagnostic probe torecognize pathological aggregated seeds of the protein, α-synuclein indiseases such as Parkinson's disease, dementia with Lewy bodies andmultiple system atrophy. In this context, embodiments of the inventioninclude methods of observing aggregated seeds of the protein,α-synuclein in a biological sample, comprising contacting the biologicalsample with a peptide that binds to residues 68-78 of α-synuclein, andthen observing if the peptide binds to aggregated seeds of the protein,α-synuclein, if present in that biological sample. Typically in thesemethods, a heterologous peptide tag is coupled to the inhibitory peptidein order to facilitate observation of the peptide. Optionally in thesemethods, the biological sample is from an individual suspected ofsuffering from Parkinson's disease, dementia with Lewy bodies ormultiple system atrophy.

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. For example, inthe preceding case, the pharmaceutical composition may comprise one ormore inhibitory peptide molecules of the invention, which can be thesame or different.

Another aspect of the invention is a complex comprising an α-synucleinmolecule (e.g. a monomer, small aggregate, oligomer, or fibril ofα-synuclein) and an inhibitory peptide of the invention. They may bebound to, conjugated with, or otherwise associated with each other. Theα-synuclein and the inhibitory peptide may be covalently ornon-covalently linked.

Other aspects of the invention include a polynucleotide encoding aninhibitory peptide of the invention; an expression vector comprising thepolynucleotide; a cell transfected with the polynucleotide or theexpression vector; and a method for making the peptide, comprisingexpressing it in the transfected cell, cultivating the cell andharvesting the peptide thus generated.

Another aspect of the invention is a method for inhibiting (preventing,stopping) aggregation of an α-synuclein molecule (e.g. a monomer, smallaggregate, oligomer, or fibril of α-synuclein), comprising contactingthe α-synuclein molecule with an effective amount of an inhibitorypeptide or pharmaceutical composition of the invention. The α-synucleinmolecule can be in solution or in a cell, which is in culture or in asubject. In one embodiment, the contacting of an α-synuclein moleculewhich is a monomer, oligomer or small aggregate prevents aggregation(oligomerization, further oligomerization, and/or fibril formation) ofthe α-synuclein molecule. In another embodiment, the contacting of anaggregated form of α-synuclein or a fibril prevents further aggregation(fibrillization) of the aggregated form or the fibril.

In one embodiment of this method, the α-synuclein protein molecule whichis contacted is in a subject having a disease or condition which ismediated by the presence of fibrillated α-synuclein (sometimes referredto herein as an α-synuclein-mediated disease or condition, or asynucleinopathy) such as, e.g., Parkinson's disease (PD), Lewy bodydementia and multiple system atrophy. α-synuclein pathologies are alsofound in other related neurodegenerative diseases, such as, e.g., bothsporadic and familial Alzheimer's disease.

Another aspect of the invention is a method for treating a subjecthaving an α-syn-mediated disease or condition, such as, e.g.,Parkinson's disease, Lewy body dementia, or multi system atrophy,comprising administering to the subject an effective amount of aninhibitory peptide or pharmaceutical composition of the invention. Thetreatment can result in the blockage (prevention) or inhibition ofα-synuclein aggregation and/or α-synuclein cytotoxicity in the subject,and spread of pathology (seeding).

Another aspect of the invention is a computer-implemented method foridentifying a peptide that inhibits α-synuclein aggregation and/orα-synuclein cytotoxicity, as described herein. Another aspect of theinvention is a kit comprising an inhibitory peptide of the invention,optionally packaged in a container. Another aspect of the invention is amethod for making an inhibitory peptide of the invention, comprisingsynthesizing it chemically or producing it recombinantly.

Yet another embodiment of the invention is a method of observing thepresence or absence of α-synuclein amyloid fibrils in a biologicalsample comprising combining a biological sample with a peptide thatbinds to residues 68-78 of α-synuclein, allowing the peptide to bind toα-synuclein amyloid fibrils that may be present in the biologicalsample, and then monitoring this combination for the presence ofcomplexes formed between α-synuclein amyloid fibrils and the peptide;wherein the presence of said complexes show the presence of α-synucleinamyloid fibrils in the biological sample. Optionally in this method, thepresence of complexes formed between α-synuclein amyloid fibrils and thepeptide is monitored using a detectable label that is coupled to thepeptide (e.g. a heterologous peptide tag). In illustrative embodimentsof the invention, the peptide comprises the sequence GAVVWGVTAVKK (SEQID NO: 3) or RAVVTGVTAVAE (SEQ ID NO: 4). Typically, the method isperformed on a biological sample obtained from an individual suspectedof suffering from Parkinson's disease. Such embodiments of the inventioncan be used, for example, in diagnostic methods designed to observe thepresence or status of PD, for example to detect disease beginningsbefore clinical symptoms, and to follow the effectiveness (or lack ofeffectiveness), of a therapeutic treatment.

Peptide inhibitors of the invention bind specifically (selectively,preferentially) to α-synuclein rather than to unintended proteins. Theprotein to which the peptide inhibitor binds may be, e.g., a monomer,small aggregate, oligomer, or fibril. For example, the binding can be 2times, 5 times, 10 times, 100 times or 200 times stronger, or no bindingat all can be detected to an unintended target. Conventional methods canbe used to determine the specificity of binding, such as e.g.competitive binding assays or other suitable analytic methods.

Active variants of the inhibitory peptides described above are alsoincluded. An “active variant” is a variant which retains at least one ofthe properties of the inhibitory peptides described herein (e.g., theability to bind to α-synuclein and/or to block, inhibit or preventα-synuclein fibrillation (aggregation) and/or α-synuclein cytotoxicity).Fibrilization, as used herein, refers to the formation of fiber orfibrils, such as amyloid fibrils.

Suitable active variants include peptidomimetic compounds (any compoundcontaining non-peptidic structural elements that is capable of mimickingthe biochemical and/or biological action(s) of a natural mimickedpeptide, including, for example, those designed to mimic the structureand/or binding activity (such as, for example, hydrogen bonds andhydrophobic packing interactions) of the peptides according to themethods disclosed herein). Inhibitory peptides of the invention,including active variants thereof, are sometimes referred to herein as“peptidic compounds” or “compounds.”

In one embodiment, active variants of the inhibitory peptides areshortened by 1-3 (e.g., 1, 2 or 3) amino acids at either the N-terminus,the C-terminus, or both of the starting inhibitory peptide. In anotherembodiment, the active variants are lengthened (extended) by 1, 2, 3 or4 amino acids at the C-terminal end of the starting inhibitory peptide,e.g. with amino acid residues at the position in which they occur inα-synuclein.

A variety of other types of active variants are included. In someembodiments, amino acids other than the ones noted above aresubstituted. These amino acids can help protect the peptide inhibitorsagainst proteolysis or otherwise stabilize the peptides, and/orcontribute to desirable pharmacodynamic properties in other ways. Insome embodiments, the non-natural amino acids allow an inhibitor to bindmore tightly to the target because the side chains optimize hydrogenbonding and/or apolar interactions with it. In addition, non-naturalamino acids offer the opportunity of introducing detectable markers,such as strongly fluorescent markers which can be used, e.g., to measurevalues such as inhibition constants. Also included are peptide mimetics,such as, e.g., peptoids, beta amino acids, N-ethylated amino acids, andsmall molecule mimetics.

In one embodiment, non-natural amino acids are substituted for aminoacids in the sequence. More than 100 non-natural amino acids arecommercially available. These include, for example,

Non-natural amino acids which can substitute for LEU:L-cyclohexylglycine 161321 - 36 - 4 L-phenylglycine 102410 - 65 - 14-hydroxy-D-phenylglycine 178119-93-2 L-α-t-butylglycine 132684 - 60 - 7cyclopentyl-Gly-OH 220497 - 61 - 0\ L-2-indanylglycine 205526 - 39 - 2Non-natural amino acids which can substitute for THR: Thr(tBu)-OH71989-35-0 (RS)-2-amino-3-hydroxy-3- 105504 - 72 - 1 methylbutanoic acidNon-natural amino acids which can substitute for ILE: allo-Ile-OH251316-98-0 N-Me-allo-Ile-OH 136092-80-3 Homoleu-OH 180414-94-2Non-natural amino acids which can substitute for ARG:Nω-nitro-L-arginine 58111-94-7 L-citrulline 133174 - 15 - 9 Non-naturalamino acids which can substitute for TYR: 3-amino-L-tyrosine 726181-70-03-nitro-L-tyrosine 136590 - 09 - 5 3-methoxy-L-tyrosine3-iodo-L-tyrosine 134486 - 00 - 3 3-chloro-L-tyrosine 478183-58-33,5-dibrimo-L-tyrosine 201484-26-6 Non-natural amino acids which cansubstitute for LYS: Lys(retro-Abz-)-OH 159322-59-5 Lys(Mca)-OH386213-32-7 (Nδ-4-methyltrityl)-L- 343770-23-0 ornithineN-α--N-ε-(d-Biotin)-L- 146987 - 10 - 2 lysine

In another embodiment, one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16 or 17) of the L-amino acids are substituted witha D amino acid.

In another embodiment, one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16 or 17)N-methylated residues are included in thepeptide.

An inhibitory peptide of the invention can comprise, e.g., L-aminoacids, D-amino acids, non-natural amino acids, or combinations thereof.

Active variants include molecules comprising various tags at theN-terminus or the C-terminus of the peptide. For example, an inhibitorypeptide of the invention can comprise as tags at its N-terminus and/orat its C-terminus: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more Lysineresidues; 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more Arginine residues; 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 or more Glutamate residues; 1, 2, 3, 4, 5,6, 7, 8, 9 or 10 or more Aspartate residues; combinations of these aminoacid residues; or other polar tags that will be evident to a skilledworker. Other active variants include mutations of the α-synucleinsequence which increase affinity of the inhibitory peptides for theα-synuclein.

In one embodiment, the inhibitor is a small molecule which has beendesigned by the methods described by Jiang et al. ⁶³ (which isincorporated herein by reference, particularly with regard to thismethod), using the atomic structure of the fiber forming segment ofα-synuclein described herein as the basis for designing the inhibitor.Suitable small molecules that can be identified by this method of Jianget al. will be evident to a skilled worker.

In one embodiment of the invention, a peptide of the invention ismodified so that 1, 2 or 3 of its amino acids are substituted with anamino acid having a non-naturally occurring side chain, such as thenon-natural amino acids discussed above, or with an amino acid having aside chain modified by cross-linking (e.g., through the epsilon aminogroup of a Lys residue) of a small molecule which has been designed byJiang et al. ⁶³. Some representative fiber-binding molecules are shownbelow. These active variants not only cap growing aggregates ofα-synuclein but also, via the modified side chains, may bind to (clampagainst) the sides of the steric zipper, thereby enhancing theinhibitory activity of the peptide.

Fiber-binding compounds designed by Jiang et al. ⁶³ include:

In one embodiment of the invention, an inhibitory peptide of theinvention is isolated or purified, using conventional techniques such asthe methods described herein. By “isolated” is meant separated fromcomponents with which it is normally associated, e.g., componentspresent after the peptide is synthesized. An isolated peptide can be acleavage product of a protein which contains the peptide sequence. A“purified” inhibitory peptide can be, e.g., greater than 90%, 95%, 98%or 99% pure.

In one embodiment, to enhance the cell permeability of an inhibitorypeptide of the invention, the peptide is fused to any of a variety ofcell penetrating peptides (CPPs). CPPs typically have an amino acidcomposition that either contains a high relative abundance of positivelycharged amino acids such as lysine or arginine or has sequences thatcontain an alternating pattern of polar/charged amino acids andnon-polar, hydrophobic amino acids. These two types of structures arereferred to as polycationic or amphipathic, respectively. A third classof CPP's are the hydrophobic peptides, containing only apolar residues,with low net charge or have hydrophobic amino acid groups that arecrucial for cellular uptake. Some typical CPP's that can be fused to aninhibitory peptide of the invention are provided in Table 2.

TABLE 2 Name Sequence Reference - original or reviewpoly ARG nR where 4 < n < 17 (e.g., n = 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16)(SEQ ID NO: 8)Wender, P.A., Mitchell, D.J., Pattabiraman, K., Pelkey, E.T., Steinman, L., andRothbard, J.B. (2000). The design, synthesis, and evaluation of molecules that enableor enhance cellular uptake: peptoid molecular transporters. Proc. Natl. Acad. Sci. U.S. A. 97, 13003-8.polyLYS nK where 4 < K < 17 (e.g., K = 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16)D-polyARG nR where 4 < n < 17 (e.g., n = 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16)D-polyLYSnK where 4 < K < 17 (e.g., K = 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16)SynB1 RGGRLSYSRRRFSTSTGR (SEQ ID NO: 9) SynB3 RRLSYSRRRF (SEQ ID NO: 10)Penetratin RQIKIWFQNRRMKWKK (SEQ ID NO: 11)Derossi, D., Joliot, A.H., Chassaing, G., and Prochiantz, A. (1994). The third helix ofthe Antennapedia homeodomain translocates through biological mem-branes. J. Biol.Chem. 269, 10444-50. Pen Arg RQIRIWFQNRRMRWRR (SEQ ID NO: 12) PenLysKQIKIWFQNKKMKWKK (SEQ ID NO: 13) TatP59W GRKKRRQRRRPWQ (SEQ ID NO: 14)Tat (48-60) GRKKRRQRRRPPQ (SEQ ID NO: 15)Vives, E., Brodin, P., and Lebleu, B. (1997). A truncated HIV-1 Tat protein basicdomain rapidly translocates through the plasma membrane and accumulates in the cellnucleus. J. Biol. Chem. 272, 16010-7. R9-TatGRRRRRRRRRPPQ (SEQ ID NO: 16)Futaki, S. (2002) Arginine-rich peptides: potential for intracellular delivery ofmacromolecules and the mystery of the translocation mechanisms. Int. J. Pharm. 245,1-7. Tat YGRKKRRQRRR (SEQ ID NO: 17)Vives, E., Brodin, P., and Lebleu, B. (1997). A truncated HIV-1 Tat protein basicdomain rapidly translocates through the plasma membrane and accumulates in the cellnucleus. J. Biol. Chem. 272, 16010-7. D-TatGRKKRRQRRRPPQ (SEQ ID NO: 18)Futaki, S. (2002) Arginine-rich peptides: potential for intracellular delivery ofmacromolecules and the mystery of the translocation mechanisms. Int. J. Pharm. 245,1-7. BMVGag(7-25) KMTRAQRRAAARRNRWTAR (SEQ ID NO: 19)Futaki, S. (2002) Arginine-rich peptides: potential for intracellular delivery ofmacromolecules and the mystery of the translocation mechanisms. Int. J. Pharm. 245,151-7. FHVCoat(35-49) RRRRNRTRRNRRRVR (SEQ ID NO: 20)Futaki, S. (2002) Arginine-rich peptides: potential for intracellular delivery ofmacromolecules and the mystery of the translocation mechanisms. Int. J. Pharm. 245,1-7. HTL V-II Rex(4-16) TRRQRTRRARRNR (SEQ ID NO: 21)Futaki, S. (2002) Arginine-rich peptides: potential for intracellular delivery ofmacromolecules and the mystery of the translocation mechanisms. Int. J. Pharm. 245,1-7. P22N-(14-30) NAKTRRHERRRKLAIER (SEQ ID NO: 22) pVECLLIILRRRIRKQAHAHSK (SEQ ID NO: 23)Elmquist, A., Lindgren, M., Bartfai, T., and Langel, Ü. (2001). VE-cadherin-derivedcell-penetrating peptide, pVEC, with carrier functions. Exp. Cell Res. 269, 237-44.Transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 24)Pooga, M., Hällbrink, M., Zorko, M., and Langel, Ü. (1998). Cell penetration bytransportan. FASEB J. 12, 67-77. TP10AGYLLGKINLKALAALAKKIL (SEQ ID NO: 25)Soomets, U., Lindgren, M., Gallet, X., Hällbrink, M., Elmquist, A., Balaspiri, L.,Zorko, M., Pooga, M., Brasseur, R., and Langel, Ü. (2000). Dele-tion analogues oftransportan. Biochim. Biophys. Acta 1467, 165-76. PTD-4PIRRRKKLRRLK (SEQ ID NO: 26) PTD-5 RRQRRTSKLMKR (SEQ ID NO: 27) Pep-1ac-KETWWETWWTEWSQPKKKRKV-cya (SEQ ID NO: 28) Pep-2ac-KETWFETWFTEWSQPKKKRKV-cya (SEQ ID NO: 29)Morris, M.C., Chaloin, L., Choob, M., Archdeacon, J., Heitz, F. and Divita, G. (2004).Combination of a new generation of PNAs with a peptide-based carrier enablesefficient targeting of cell cycle progression. Gene Ther 11: 757-764.Pep-3 ac-KWFETWFTEWPKKRK-cya (SEQ ID NO: 30)Morris, M.C., Gros, E., Aldrian-Herrada, G., Choob, M., Archdeacon, J., Heitz, F. et al.(2007). A non-covalent peptide-based carrier for in vivo delivery of DNA mimics.Nucleic Acids Res 35: e49. E N(1-22)MDAQTRRRERRAEKQAQWKAAN (SEQ ID NO: 31) B 21 N-(12-29)TAKTRYKARRAELIAERR (SEQ ID NO: 32) U2AF(142-153)SQMTRQARRLYV (SEQ ID NO: 33) PRP6(129-144)TRRNKRNRIQEQLNRK (SEQ ID NO: 34) MAP KLALKLALKLALALKLA (SEQ ID NO: 35)SBP MGLGLHLLVLAAALQGAWSQPKKKRKV (SEQ ID NO: 36) FBPGALFLGWLGAAGSTMGAWSQPKKKRKV (SEQ ID NO: 37) MPGac-GALFLGFLGAAGSTMGAWSQPKKKRKV-cya (SEQ ID NO: 38)Morris, M.C., Vidal, P., Chaloin, L., Heitz, F. and Divita, G. (1997). A new peptidevector for efficient delivery of oligonucleotides into mammalian cells. Nucleic AcidsRes 25: 2730-2736. MPG(ΔNLS)ac- GALFLGFLGAAGSTMGAWSQPKSKRKV-cya (SEQ ID NO: 39) REV(34-50)TRQARRNRRRRWRERQR (SEQ ID NO: 40)Futaki, S. (2002) Arginine-rich peptides: potential for intracellular delivery ofmacromolecules and the mystery of the translocation mechanisms. Int. J. Pharm. 245,1-7.ACPPs from Jiang et al., PNAS 2004 - lower case indicates D-aa. The symbol “_” insome of these sequences indicates a position at which any of a variety of art-recognized protease cleavage sites can be inserted:EEEEEDDDDK_AXRRRRRRRRRXC (SEQ ID NO: 41)EEEEEDDDDK_ARRRRRRRRRXC (SEQ ID NO: 42)EDDDDK_AXRRRRRRRRRXC (SEQ ID NO: 43)EEDDDDK_ARXRRXRRXRRXRRXC (SEQ ID NO: 44)DDDDDDK_ARRRRRRRRRXC (SEQ ID NO: 45)

In another embodiment, the CPP is polyD(₁₋₁₆).

In general, it is advisable that the length of the CPP is rather short,e.g. less than about 30 amino acids, in order to improve stability andpharmacodynamic properties once the molecule enters a cell.

In some embodiments, the CPP is directly attached (fused) to a peptideof the invention. In other embodiments, it is desirable to separate thehighly charged CPP from the inhibitor peptide with a linker, to allowthe inhibitor to retain its activity. Any of a variety of linkers can beused. The size of the linker can range, e.g., from 1-7 or even moreamino acids (e.g., 1, 2, 3, 4, 5, 6 or 7 amino acids).For example, thelinker can be QVTNVG at the N-terminus and QKTVEG at the C-terminus or atruncated version thereof having 1, 2, 3, 4 or 5 of the contiguous aminoacids N-terminal to to the inhibitory peptide.

In embodiments of the invention, the inhibitory peptide is detectablylabeled. Labeled peptides can be used, e.g., to better understand themechanism of action and/or the cellular location of the inhibitorypeptide. Suitable labels which enable detection (e.g., provide adetectable signal, or can be detected) are conventional and well-knownto those of skill in the art. Suitable detectable labels include, e.g.,radioactive active agents, fluorescent labels, and the like. Methods forattaching such labels to a protein, or assays for detecting theirpresence and/or amount, are conventional and well-known.

An inhibitory peptide of the invention can be synthesized (e.g.,chemically or by recombinant expression in a suitable host cell) by anyof a variety of art-recognized methods. In order to generate sufficientquantities of an inhibitory peptide for use in a method of theinvention, a practitioner can, for example, using conventionaltechniques, generate nucleic acid (e.g., DNA) encoding the peptide andinsert it into an expression vector, in which the sequence is under thecontrol of an expression control sequence such as a promoter or anenhancer, which can then direct the synthesis of the peptide. Forexample, one can (a) synthesize the DNA de novo, with suitable linkersat the ends to clone it into the vector; (b) clone the entire DNAsequence into the vector; or (c) starting with overlappingoligonucleotides, join them by conventional PCR-based gene synthesismethods and insert the resulting DNA into the vector. Suitableexpression vectors (e.g., plasmid vectors, viral, including phage,vectors, artificial vectors, yeast vectors, eukaryiotic vectors, etc.)will be evident to skilled workers, as will methods for making thevectors, inserting sequences of interest, expressing the proteinsencoded by the nucleic acid, and isolating or purifying the expressedproteins.

Another aspect of the invention is a pharmaceutical compositioncomprising one or more of the inhibitory peptides and a pharmaceuticallyacceptable carrier. The components of the pharmaceutical composition maybe detectably labeled, e.g. with a radioactive or fluorescent label, orwith a label, for example one that is suitable for detection by positronemission spectroscopy (PET) or magnetic resonance imaging (MRI). Forexample, peptides of the invention can be coupled to a detectable labelselected from the group consisting of a radioactive label, aradio-opaque label, a fluorescent dye, a fluorescent protein, acolorimetric label, and the like. In some embodiments, the inhibitorypeptide is present in an effective amount for the desired purpose.

“Pharmaceutically acceptable” means that which is useful in preparing apharmaceutical composition that is generally safe, non-toxic, andneither biologically nor otherwise undesirable and includes that whichis acceptable for veterinary as well as human pharmaceutical use. Forexample, “pharmaceutically acceptable salts” of a compound means saltsthat are pharmaceutically acceptable, as defined herein, and thatpossess the desired pharmacological activity of the parent compound.

Another aspect of the invention is a polynucleotide encoding aninhibitory peptide of the invention. In embodiments of the invention,the polynucleotide is operably linked to a regulatory control sequence(e.g., a promoter or an enhancer) to facilitate production of theencoded protein following introduction (e.g. by transfection) into asuitable cell. Other embodiments include a cell comprising theexpression vector; and a method of making an inhibitory peptide of theinvention comprising cultivating the cell and harvesting the peptidethus generated.

As used throughout this application, “about” means plus or minus 5% of avalue.

Another aspect of the invention is a kit for carrying out any of themethods described herein. The kit may comprise a suitable amount of aninhibitory peptide of the invention; reagents for generating thepeptide; reagents for assays to measure their functions or activities;or the like. Kits of the invention may comprise instructions forperforming a method. Other optional elements of a kit of the inventioninclude suitable buffers, media components, or the like; a computer orcomputer-readable medium providing the structural representation of acrystal structure described herein; containers; or packaging materials.Reagents for performing suitable controls may also be included. Thereagents of the kit may be in containers in which the reagents arestable, e.g., in lyophilized form or stabilized liquids. The reagentsmay also be in single use form, e.g., in single reaction form foradministering to a subject.

Characterization of candidate inhibitory peptides of the invention canbe carried out by any of a variety of conventional methods. For example,the peptides can be assayed for the ability to reduce or inhibitα-synuclein aggregation or cytotoxicity or cell-to-cell spread. Theassays can be carried out in vitro or in vivo. Suitable assays will beevident to a skilled worker; some suitable assays are described herein.

One aspect of the invention is a method for reducing or inhibitingα-synuclein aggregation, comprising contacting α-synucleinprotofilaments with an effective amount of one or more of the inhibitorypeptides of the invention. Such a method can be carried out in solutionor in a cell (e.g. cells in culture or in a subject).

Another aspect of the invention is a method for treating a subjecthaving a disease or condition which is mediated by the presence offibrillated α-synuclein (sometimes referred to herein as anα-synuclein-mediated disease or condition), comprising administering tothe subject an effective amount of an inhibitory peptide orpharmaceutical composition of the invention. Among such diseases orconditions are, e.g., Parkinson's disease (PD), Lewy body dementia, ormultiple system atrophy. Another aspect of the invention is a method toprevent the onset of such diseases or conditions (e.g., PD), or to treata subject in the early stages of such diseases or conditions, or that isdeveloping such a disease or condition, in order to prevent or inhibitdevelopment of the condition or disease.

An inhibitory peptide or pharmaceutical composition of the invention issometimes referred to herein as an “inhibitor.”

An “effective amount” of an inhibitor of the invention is an amount thatcan elicit a measurable amount of a desired outcome, e.g. inhibition ofα-synuclein aggregation or cytotoxicity; for a diagnostic assay, anamount that can detect a target of interest, such as an α-synucleinaggregate; or in a method of treatment, an amount that can reduce orameliorate, by a measurable amount, a symptom of the disease orcondition that is being treated.

A “subject” can be any subject (patient) having aggregated (fibrillated)α-synuclein molecules associated with a condition or disease which canbe treated by a method of the present invention. In one embodiment ofthe invention, the subject has PD. Typical subjects include vertebrates,such as mammals, including laboratory animals, dogs, cats, non-humanprimates and humans.

The inhibitors of the invention can be formulated as pharmaceuticalcompositions in a variety of forms adapted to the chosen route ofadministration, for example, orally, nasally, intraperitoneally, orparenterally, by intravenous, intramuscular, topical or subcutaneousroutes, or by injection into tissue.

Suitable oral forms for administering the inhibitors include lozenges,troches, tablets, capsules, effervescent tablets, orally disintegratingtablets, floating tablets designed to increase gastric retention times,buccal patches, and sublingual tablets.

The inhibitors of the invention may be systemically administered, e.g.,orally, in combination with a pharmaceutically acceptable vehicle suchas an inert diluent or an assimilable edible carrier, or by inhalationor insufflation. They may be enclosed in coated or uncoated hard or softshell gelatin capsules, may be compressed into tablets, or may beincorporated directly with the food of the patient's diet. For oraltherapeutic administration, the compounds may be combined with one ormore excipients and used in the form of ingestible tablets, buccaltablets, troches, capsules, elixirs, suspensions, syrups, wafers, andthe like. For compositions suitable for administration to humans, theterm “excipient” is meant to include, but is not limited to, thoseingredients described in Remington: The Science and Practice ofPharmacy, Lippincott Williams & Wilkins, 21st ed. (2006) (hereinafterRemington's).

The inhibitors may be combined with a fine inert powdered carrier andinhaled by the subject or insufflated. Such compositions andpreparations should contain at least 0.1% compounds. The percentage ofthe compositions and preparations may, of course, be varied and mayconveniently be between about 2% to about 60% of the weight of a givenunit dosage form.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. A syrup or elixir may contain the activecompound, sucrose or fructose as a sweetening agent, methyl andpropylparabens as preservatives, a dye and flavoring such as cherry ororange flavor.

Various other materials may be present as coatings or to otherwisemodify the physical form of the solid unit dosage form. For instance,tablets, pills, or capsules may be coated with gelatin, wax, shellac orsugar and the like. Of course, any material used in preparing any unitdosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed.

In addition, the inhibitors may be incorporated into sustained-releasepreparations and devices. For example, the inhibitors may beincorporated into time release capsules, time release tablets, and timerelease pills. In some embodiments, the composition is administeredusing a dosage form selected from the group consisting of effervescenttablets, orally disintegrating tablets, floating tablets designed toincrease gastric retention times, buccal patches, and sublingualtablets.

The inhibitors may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the inhibitorscan be prepared in water, optionally mixed with a nontoxic surfactant.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, triacetin, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations can contain apreservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the compounds which are adapted for the extemporaneouspreparation of sterile injectable or infusible solutions or dispersions,optionally encapsulated in liposomes. In all cases, the ultimate dosageform should be sterile, fluid and stable under the conditions ofmanufacture and storage. The liquid carrier or vehicle can be a solventor liquid dispersion medium comprising, for example, water, ethanol, apolyol (for example, glycerol, propylene glycol, liquid polyethyleneglycols, and the like), vegetable oils, nontoxic glyceryl esters, andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the formation of liposomes, by the maintenance of therequired particle size in the case of dispersions or by the use ofsurfactants.

Sterile injectable solutions are prepared by incorporating the compoundsin the required amount in the appropriate solvent with various of theother ingredients enumerated above, as required, followed by filtersterilization. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze drying techniques, which yield a powder of theactive ingredient plus any additional desired ingredient present in thepreviously sterile-filtered solutions.

For topical administration, the compounds may be applied in pure form.However, it will generally be desirable to administer them to the skinas compositions or formulations, in combination with a dermatologicallyacceptable carrier, which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Other solidcarriers include conventional nontoxic polymeric nanoparticles ormicroparticles. Useful liquid carriers include water, alcohols orglycols or water/alcohol/glycol blends, in which the compounds can bedissolved or dispersed at effective levels, optionally with the aid ofnon-toxic surfactants. Adjuvants such as fragrances and additionalantimicrobial agents can be added to optimize the properties for a givenuse. The resultant liquid compositions can be applied from absorbentpads, used to impregnate bandages and other dressings, or sprayed ontothe affected area using pump-type or aerosol sprayers.

Useful dosages of the peptides or pharmaceutical compositions of theinvention can be determined by comparing their in vitro activity, and invivo activity in animal models. Methods for the extrapolation ofeffective dosages in mice, and other animals, to humans are known to theart.

For example, the concentration of the compounds in a liquid composition,such as a lotion, can be from about 0.1-25% by weight, or from about0.5-10% by weight. The concentration in a semi-solid or solidcomposition such as a gel or a powder can be about 0.1-5% by weight, orabout 0.5-2.5% by weight.

Effective dosages and routes of administration of agents of theinvention are conventional. The exact amount (effective dose) of theagent will vary from subject to subject, depending on, for example, thespecies, age, weight and general or clinical condition of the subject,the severity or mechanism of any disorder being treated, the particularagent or vehicle used, the method and scheduling of administration, andthe like. A therapeutically effective dose can be determinedempirically, by conventional procedures known to those of skill in theart. See, e.g, The Pharmacological Basis of Therapeutics, Goodman andGilman, eds., Macmillan Publishing Co., New York. For example, an,effective dose can be estimated initially either in cell culture assaysor in suitable animal models. The animal model may also be used todetermine the appropriate concentration ranges and routes ofadministration. Such information can then be used to determine usefuldoses and routes for administration in humans. A therapeutic dose canalso be selected by analogy to dosages for comparable therapeuticagents.

The particular mode of administration and the dosage regimen will beselected by the attending clinician, taking into account the particularsof the case (e.g., the subject, the disease, the disease state involved,and whether the treatment is prophylactic). Treatment may involve dailyor multi-daily doses of compound(s) over a period of a few days tomonths, or even years.

In general, however, a suitable dose will be in the range of from about0.001 to about 100 mg/kg, e.g., from about 0.01 to about 100 mg/kg ofbody weight per day, such as above about 0.1 mg per kilogram, or in arange of from about 1 to about 10 mg per kilogram body weight of therecipient per day. For example, a suitable dose may be about 1 mg/kg, 5mg/kg, 10 mg/kg, 20 mg/kg, or 30 mg/kg of body weight per day.

The inhibitors are conveniently administered in unit dosage form; forexample, containing 0.05 to 10000 mg, 0.5 to 10000 mg, 5 to 1000 mg, orabout 100 mg of active ingredient per unit dosage form. In someembodiments, the dosage unit contains about 0.1 mg, about 0.5 mg, about1 mg, about 10 mg, about 25 mg, about 50 mg, about 75 mg, or about 100mg, of active ingredient.

EXAMPLES Example I—the Toxic Core of α-Synuclein of Parkinson's Disease:Structure from Invisible Crystals

We carried out screens for crystals of peptide segments within the NACdomain and adjacent regions, seeking structural information on themolecular basis of aggregation and toxicity of α-syn.

Extensive crystal screens of two segments, NACore, residues⁶⁸GAVVTGVTAVA⁷⁸ (SEQ ID NO:46), and PreNAC, ⁴⁷GVVHGVTTVA⁵⁶ (SEQ IDNO:47), seemingly produced non-crystalline, amorphous aggregates. But onexamination by electron microscopy, we found the aggregates to beclusters of elongated nanocrystals only 50-300 nm in cross section andthus invisible by conventional light microcopy. We confirmedwell-ordered crystallinity of NACore at both the SACLA and LCLS freeelectron lasers. We also found that a 9-residue fragment within theNACore, which we term SubNACore, ⁶⁹AVVTGVTAV⁷⁷ (SEQ ID NO:48), yieldedcrystals 1,000-10,000 times larger in volume than the NACorenanocrystals. We were therefore able to apply synchrotronmethods^(18,19) to these larger crystals to determine the structure ofthe amyloid-like fibrils. Although this 9-residue fragment is missingonly two residues compared with NACore, it is not as toxic²⁰, offeringinsight described below, into the toxicity of α-syn.

To determine the structure of the invisible crystals of NACore andPreNAC, we turned to MicroED ²¹. In MicroED an extremely low doseelectron beam is directed on a nanocrystal within a transmissionelectron microscope under cryogenic conditions, yielding diffractionpatterns. At the wavelength used in our experiments at 200 keV is verysmall (0.025 Å) the Ewald sphere is essentially flat yieldingdiffraction patterns that closely resemble a 2D slice through 3Dreciprocal space. As the crystal is continuously rotated in the beam, aseries of such diffraction patterns is collected ⁵. Scaling togetherdiffraction data collected from multiple crystals produces a full 3Ddiffraction dataset. MicroED has been successfully applied to thewell-known structures of hen egg-white lysozyme ^(6,5), bovine livercatalase²² and Ca2+-ATPase²³. But NACore and PreNAC are the firstpreviously unknown structures determined by MicroED.

For NACore and PreNAC, we collected electron diffraction patterns fromnano-crystals that lay preferentially oriented, flat on the surface of aholey carbon Quantifoil grid, in a frozen-hydrated state. Grids werefirst screened for appropriately sized crystals, and candidate crystalsscreened for diffraction. We used crystals showing strong diffractionfor data collection by continuous unidirectional rotation about a fixedaxis, acquiring a series of diffraction frames at fixed time intervals⁵.The needle-shaped crystals typically exceeded the length needed fordiffraction; those that were unbent and 100 to 300 nm wide produced thebest diffraction patterns. Data from multiple crystals were integrated,scaled and merged together.

The multi-crystal NACore and PreNAC datasets were phased by molecularreplacement, using the atomic model of SubNACore and an ideal betastrand model, respectively, as probes. Diffraction phases calculatedfrom the SubNACore probe structure and NACore structure factors yieldeda difference density map, which clearly reveals the positions of themissing residues, after subsequent refinement, two water molecules, andseveral hydrogen atoms. Full models of NACore and PreNAC were refinedagainst the MicroED data, producing structures at 1.4 Å resolution withacceptable R-factors. Electron scattering factors were used in therefinement calculations ²⁴.

The structure of the NACore peptide chain is a nearly fully extendedβ-strand. These NACore strands stack in-register into β-sheets, as hadbeen predicted by site-directed spin labeling ^(25,26.) The sheets arepaired, as is usual in amyloid spines, and the pairs of sheets formtypical steric-zipper protofilaments, previously seen as the spines inmany amyloid-like fibrils formed from short segments of fibril-formingproteins. The unusual features of this steric zipper are that the11-residue width of the zipper is longer than we have previouslyobserved, and each pair of sheets contains two water molecules, eachassociated with a threonine sidechain, within the interface instead ofbeing completely dry. Also, in our crystals of NACore, each sheet formstwo snug interfaces: Interface A with 268 Å2 of buried accessiblesurface area per chain, is more extensive and presumably stronger thanInterface B (167 Å2), because the terminal residues of the chains inopposing sheets bend towards each other. The structure of PreNAC revealsa peptide chain that forms a β-strand kinked at residue glycine 51.These strands are arranged into pairs of β-sheets that like the NACorestructure interdigitate to form steric zipper protofilaments (FIG. 3 ).Of special note, a five residue segment of PreNAC (⁵¹GVTTV⁵⁵) (SEQ IDNO:49), differs in only one residue from a five residue segment ofNACore (⁷³GVTAV⁷⁷) (SEQ ID NO:50), and their backbones and identicalsidechains superimpose closely with an alpha carbon RMS deviation of 1.5Å. This means that weaker interface B of NACore mimics a hypotheticalinterface between NACore and PreNAC. Below we suggest the significanceof the possible contact between these two segments of α-syn.

The relevance of the structure of NACore to fibrils of full length α-synis established by the resemblance of their diffraction patterns.Specifically, the fiber diffraction pattern of aligned fibrils offull-length and N-terminally acetylated²⁷ α-syn protein display the sameprincipal peaks as the diffraction of aligned NACore nanocrystals. Allthree fibrils display the strong reflection at 2.4 Å in theirdiffraction patterns. This reflection arises in NACore because oneβ-sheet of the steric zipper is translated along the fiber axis withrespect to the other β-sheet by 2.4 Å, one half the 4.8 Å spacingbetween β-strands, permitting the two sheets to interdigitate tightlytogether. All three share a strong 4.6 Å reflection, which in NACoreresults from both the stacking of β-strands and the staggering betweenadjacent β-sheets of the steric zipper, while a shared reflection atnear 8.2 Å likely arises from the distance between the adjacent pairs ofβ-sheets that make up the α-syn fibril. This comparison of fiberdiffraction patterns strongly suggests that the structure of NACore issimilar to the spine of our toxic fibrils of full α-syn.

The combined structures of NACore and PreNAC allow us to construct aspeculative model for much of the ordered segments of the A53T earlyonset mutant α-syn. Experimental support of this model comes from theagreement of its simulated fiber diffraction with the measureddiffraction patterns of α-syn and N-acetyl α-syn fibrils as well asaligned NACore nanocrystals. Above we noted that the weaker Interface Bof NACore mimics a hypothetical interaction of PreNAC with NACore. Infact, the interacting sidechains in the weaker NACore Interface B (G73,T75, and V77) are identical to the sidechains (G51, T53, V55)interacting in the hypothetical interface of PreNAC with NACore.

The identity and structure of the cytotoxic amyloid formed by α-synremains a subject of intensive research ^(17,28,29,30,31,32). The weightof evidence over the past decade has tilted scientific opinion from thefully developed amyloid fibrils found in Lewy bodies as the toxicentities to smaller, transient amyloid oligomers. Yet recently,quantitative arguments have been put forward in favor of fibrils ³³.Without wishing to be bound by any particular mechanism, it is suggestedthat our experiments of the cytotoxicity of NACore on neuroblastomacells are consistent with the view that fibrils are toxic: we find thatNACore shaken and aggregated for 72 hours displays abundant fibrils, ismore toxic than freshly dissolved NACore and is comparably toxic tosimilarly aggregated full α-syn. We also find greater cytotoxicity ofNACore than SubNACore, which is shorter by two residues. This isconsistent with the more rapid fibril formation of NACore than ofSubNACore. These observations do not rule out the formation of anon-fibrillar, oligomeric assembly, present, but invisible, in ouraggregated samples of NACore and α-syn. Of course, NACore is merely afragment of full length α-syn, and lacks most of the membrane-bindingmotifs of the N-terminus of the protein which have been implicated inmembrane disruption ^(34,35). Yet it is clear that NACore is the minimumentity that recapitulates all the features of full length α-synaggregation and toxicity.

The miniscule size of NACore is typical of amyloid crystals and also ofvarious other biological crystals of interest. For amyloid crystals, ourspeculation is that the tiny size is a consequence of the natural twistof β-sheets that form the protofilaments of the fibrils. The crystallattice restrains the twist, creating a strain in these crystals, whichincreases as crystals grow. Eventually this strain prevents furtheraddition of β-strands, limiting the thickness of the needle crystals. Inour experience, longer segments (for example, 11 residues compared to 9residues) limit crystal growth even more; in the case of 11-residueNACore and 10-residue PreNAC, the strain produces nanocrystals,invisible by optical microscopy. These crystals are too small formounting and conventional synchrotron data collection, but are ideallysuited for analysis by MicroED. Our structures of NACore and PreNACdemonstrate that

MicroED is capable of determining new and accurate structures ofbiological material at atomic resolution. This finding paves the pathfor applications of MicroED to other biological substances ofimportance, for which only nanocrystals can be grown. In our particularapplication, we have been able to learn the atomic arrangement of thecore of the crucial NAC domain. This opens opportunities forstructure-based design of inhibitors of amyloid formation of α-syn ³⁶.

Methods Crystallization

Microcrystals of SubNACore, ⁶⁹AVVTGVTAV⁷⁷ (SEQ ID NO:48), were grownfrom synthetic peptide purchased from CS Bio. Crystals were grown atroom temperature by hanging drop vaporization. Lyophilized peptide wasdissolved in water at 2.9 mg/ml concentration in 48 mM lithiumhydroxide. Peptide was mixed in a 2:1 ratio with reservoir containing0.9 M ammonium phosphate, and 0.1M sodium acetate pH 4.6.

Nanocrystals of NACore, ⁶⁸GAVVTGVTAV⁷⁸ (SEQ ID NO:46), were grown fromsynthetic peptide purchased from CS Bio. Ten batches of synthesizedpeptide (CSBio) at a concentration of 1 mg/ml in sterile water wereshaken at 37° C. on a Torrey Pines orbital mixing plate at speed setting9, overnight. The insoluble material was washed in 30% (w/v) glycerolthen stored in water at room temperature before diffraction. The samplecontained a mixture of fibrils and crystals.

Nanocrystals of PreNAC, ⁴⁷GVVHGVTTVA⁵⁶ (SEQ ID NO:47), were grown fromsynthetic peptide purchased from InnoPep. Crystallization trials ofsynthesized peptide were prepared in batch. Peptide was weighed anddissolved in sterile-filtered 50 mM phosphate buffer pH 7.0 with 0.1%DMSO at a concentration of 5 mg/ml. This solution was shaken at 37° C.on a Torrey Pines orbital mixing plate at speed setting 9, overnight.

Data Collection and Processing

X-ray diffraction data from microcrystals of SubNACore were collectedusing synchrotron radiation at the Advanced Photon Source, NortheastCollaborative Access Team microfocus beamline 24-ID-E. The beamline wasequipped with an ADSC Quantum 315 CCD detector. Data from a singlecrystal were collected in 5° wedges at a wavelength of 0.9791 Å using a5 μm beam diameter. We used data from three different sections along theneedle axis. The crystals were cryo-cooled (100 K) for data collection.Data were processed and reduced using Denzo/Scalepack from the HKL suiteof programs³⁷.

X-ray diffraction data from nanocrystals of NACore were collected usingXFEL radiation at the CXI instrument (Coherent X-ray Imaging) at theLinear Coherent Light Source (LCLS)-SLAC. The photon energy of the X-raypulses was 8.52 keV (1.45 Å). Each 40 fs pulse contained up to 6×1011photons at the sample position taking into account a beamlinetransmission of 60%. The diameter of the beam was approximately 1 μm. Weused a concentration of approximately 25 μl of pelleted materialsuspended in 1 mL water. The sample was injected into the XFEL beamusing a liquid jet injector and a gas dynamic virtual nozzle³⁸. Themicro jet width was approximately 4 μm and the flow rate was 40 μl/min.The sample caused noticeable sputtering of the liquid jet. XFEL datawere processed using cctbx.xfel ^(39,40).

Electron diffraction data from nanocrystals of NACore and PreNAC werecollected using MicroED techniques^(5,6). These nanocrystals typicallyclump together. To break up the clumps, an approximately 100 μL volumeof nanocrystals was placed in a sonication bath for 30 minutes.Nanocrystals were deposited onto a quantifoil holey-carbon EM grid in a2-3p1 drop after appropriate dilution, which optimized for crystaldensity on the grid. All grids were then blotted and vitrified byplunging into liquid ethane using a Vitrobot Mark IV (FEI), thentransferring to liquid nitrogen for storage. Frozen hydrated grids weretransferred to a cryo-TEM using a Gatan 626 cryo-holder. Diffractionpatterns and crystal images were collected using an FEG-equipped FEITecnai F20 TEM operating at 200 kV and recorded using a bottom mountTVIPS F416 CMOS camera with a sensor size of 4000 squared pixels, each15.6 μm in size per square dimension. Diffraction patterns were recordedby operating the detector in rolling shutter mode with 2×2 pixelbinning, producing a final image 2000 squared pixels in size. Individualimage frames were taken with exposure times of 3-4 seconds per image,using a selected area aperture with an illuminating spot size ofapproximately one micron. This geometry equates to an electron dose ofless than 0.1 e−/Å2 per second. During each exposure, crystals werecontinuously rotated within the beam at a rate of 0.3° per second,corresponding to 1.2° wedge per frame. Diffraction data were collectedfrom several crystals each oriented differently with respect to therotation axis. These data sets each spanned wedges of reciprocal spaceranging from 40° to 80°.

Calibration of the sample to detector distance was accomplished using apolycrystalline gold standard and by referencing the prominentreflections in the electron diffraction experiment with thecorresponding reflections in the XFEL data. Calibration of the x/ylocations of the 64-tile CSPAD detector was performed by cctbx.xfel byrefining the optically measured tile positions against a thermolysindata set³⁹.

To gain compatibility with conventional X-ray data processing programs,the diffraction images were converted from tiff or TVIPS format to theSMV crystallographic format. We used XDS to index the diffractionimages⁴¹, and XSCALE for merging and scaling together data setsoriginating from different crystals. For NACore, data from four crystalswere merged, while for PreNAC, data from three crystals were merged toassemble the final data sets.

Structure Determination

The molecular replacement solution for SubNACore was obtained using theprogram Phaser 42. The search model consisted of a geometrically idealβ-strand composed of nine alanine residues. Crystallographic refinementswere performed with the program REFMAC ⁴³. The molecular replacementsolution for NACore was obtained using the program Phaser 42. The searchmodel consisted of the SubNACore structure determined previously.Crystallographic refinements were performed with the program Phenix ⁴⁴.The molecular replacement solution for PreNAC was obtained using theprogram Phaser ⁴². The search model consisted of a geometrically idealβ-strand composed of six residues with sequence GVTTVA. Crystallographicrefinements were performed with the program Phenix ⁴⁴.

Model building for all segments was performed using COOT ⁴⁵. Thecoordinates of the final models and the structure factors have beendeposited in the Protein Data Bank with PDB code 4RIK for SubNACore,4RIL for NACore, and 4ZNN for PreNAC. The structures were illustratedusing Pymol ⁴⁶.

α-synuclein

The human wild type α-syn construct was previously characterized⁴⁷(pRK172, ampicillin, T7 promoter) with sequence:

(SEQ ID NO: 1) MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA.α-synuclein Purification

Full length α-syn was purified according to published protocols³¹. Theα-syn construct was transformed into E. coli expression cell line BL21(DE3) gold (Agilent Technologies, Santa Clara, Calif.) for wild typeα-syn protein expression. A single colony was incubated into 100 mL LBMiller broth (Fisher Scientific, Pittsburgh, Pa.) supplemented with 100μg/mL ampicillin (Fisher Scientific, Pittsburgh, Pa.) and grownovernight at 37° C. One liter of LB Miller supplemented with 100 μg/mLampicillin in 2 L shaker flasks was incubated with 10 mL of overnightculture and grown at 37° C. until the culture reached an OD600 ˜0.6-0.8as measured by a BioPhotometer UV/VIS Photometer (Eppendorf, Westbury,N.Y.). IPTG (Isopropyl β-D-1-thiogalactopyranoside) was added to a finalconcentration of 0.5 mM, and grown for 4-6 hours at 30° C. Cells wereharvested by centrifugation at 5,500×g for 10 minutes at 4° C. The cellpellet was frozen and stored at −80° C.

The cell pellet was thawed on ice and resuspended in lysis buffer (100mM Tris-HCl pH 8.0, 500 mM NaCl, 1 mM EDTA pH 8.0) and lysed bysonication. Crude cell lysate was clarified by centrifugation at15,000×g for 30 minutes at 4° C. The clarified cell lysate was boiledand cell debris was removed by centrifugation. Protein in thesupernatant was precipitated in acid at pH 3.5 through addition of HClby titration to protein solution on ice while stirring then centrifugedfor an additional 15,000×g for 30 minutes at 4° C. Supernatant wasdialyzed against buffer A (20 mM Tris-HCl, pH 8.0). After dialysis thesolution was filtered through a 0.45 μm syringe (Corning, N.Y. 14831)before loading onto a 20 mL HiPrep Q HP 16/10 column (GE Healthcare,Piscataway, N.J.). The Q-HP column was washed with five column volumesof buffer A and protein eluted using a linear gradient to 100% in fivecolumn volumes of buffer B (20 mM Tris-HCl, 1M NaCl, pH 8.0). Proteineluted at around 50-70% buffer B; peak fractions were pooled. Pooledsamples were concentrated approximately tenfold using Amicon Ultra-15centrifugal filters. Approximately 5 ml of the concentrated sample wasloaded onto a HiPrep 26/60 Sephacryl S-75 HR column equilibrated withfiltration buffer (25 mM sodium phosphate, 100 mM NaCl, pH 7.5). Peakfractions were pooled from the gel filtration column and dialyzedagainst 5 mM Tris-HCl, pH 7.5, concentrated to 3 mg/ml. These werefiltered through a 0.2 μm pore size filter (Corning, N.Y. 14831) andstored at 4° C.

Recombinantly expressed full-length α-syn with an N-terminal acetylationwas prepared and purified in the following way based on a protocol byDer-Sarkissian et al²⁵

The α-syn plasmid was co-expressed with a heterodimeric proteinacetylation complex from S. pombe to acetylate the N-terminus(pACYC-DUET, chloramphenicol, T7 promoter)⁴⁸. The two vectors wereco-transformed into E. coli BL21 (DE3) using media containing bothampicillin and chloramphenicol. Cell cultures were grown in TB mediacontaining ampicillin and chloramphenicol and induced to express α-synwith 0.5 mM IPTG overnight at 25° C. Cells were harvested bycentrifugation, the cell pellet then resuspended in lysis buffer (100 mMTris-HCl pH 8.0, 500 mM NaCl, 1 mM EDTA pH 8.0, and 1 mMphenylmethylsulfonyl fluoride) and cells lysed using an Emulsiflexhomogenizer (Avestin). The lysate was boiled and debris removed bycentrifugation. A protein fraction was also removed by precipitation atlow pH on ice followed by centrifugation. The remaining supernatant waspH adjusted by titration and dialyzed against Buffer A (20 mM Tris-HCl,pH 8.0, 1 mM DTT, 1 mM EDTA, pH 8.0). The resulting protein solution wasloaded onto a 5 mL Q-Sepharose FF column (GE Healthcare) equilibratedwith Buffer A and eluted against a linear gradient of Buffer B (1M NaCl,20 mM Tris-HCl, pH 8.0, 1 mM DTT, 1 mM EDTA, pH 8.0). Fractionscontaining α-syn were identified using SDS-PAGE, collected, concentratedand further purified by size exclusion (Sephacryl S-100 16/60, GEHealthcare) in 20 mM Tris pH 8.0, 100 mM NaCl, 1 mM DTT, 1 mM EDTA.Purity of fractions was assessed by SDS-PAGE.

Acetylated protein was characterized by LC-MS27,⁴⁹. Expected averagemass: 14460.1 Da for alpha-synuclein and 14502.1 for acetylatedalpha-synuclein. Observed average mass: 14464.0 Da for alpha-synucleinand 14506.0 for acetylated alpha-synuclein. The shift of 4 Da betweenobserved and expected average masses is due to instrumental error.

Inhibitor Synthesis

Inhibitors were commercially obtained from Genscript Inc. at greaterthan 98% purity and solubilized in 100% DMSO at 10 mM concentration.Solubilized inhibitors were filtered with 0.1 μM filter and stored at−20° C. in 20 μL aliquots until further use.

Thioflavin T Assays

Fibril formation assays were performed with 50 μM protein concentrationin conditions identical to those used for aggregating proteins forseeding assay, but with the addition of ThT. All assays were carried outin black Nunc 96-well optical bottom plates (Thermo Scientific). Plateswere agitated at 600 rpm in 3-mm rotation diameter in a Varioskanmicroplate reader (Thermo) at 37° C. Fluorescence measurements wererecorded every 30 mins using λ_(ex)=444 nm, λ_(em)=482 nm, with anintegration time of 200 μs.

Aggregation Assays

Purified α-synuclein was dialyzed in 0.1 M sodium sulfate, 25 mM sodiumphosphate and aggregated by shaking in Torrey Pine shakers at 50 μM at37° C. at speed 9 for 5-6 days.

Extraction of Sarkosyl Insoluble Protein Filaments from PD Human BrainTissues

Human frozen brain tissues were obtained from UCLA Brain TumorTranslational Resource (BTTR). Sarkosyl insoluble protein was extractedusing previously described protocols. Briefly, frozen tissue washomogenized in ice cold PBS using a dounce homogenizer at 200 mg/mL. Thehomogenate was then diluted in buffer A (10 mM Tris 7.4, 800 mM NaCl, 1mM EGTA, 10% Sucrose) to 50 mg/mL in total volume of 1 mL andcentrifuged at 20,700 g for 20 min at 4° C. The supernatant wascollected in an ultracentrifuge tube and pellet was resuspended in 0.5mL buffer A followed by centrifugation at 20,700 g for 20 mins at 4° C.The supernatants pooled together. 150 μl 10% sarkosyl (w/v) in Milliporewater was then added and incubated for 1 h at room temperature on flatrotating shaker at 700 rpm. The solution was then centrifuged at 100,000g for 1 h at 4° C. using SW 55 Rotor (Beckman Coulter). The supernatanatwas discarded and pellet was washed with 5 mL Buffer A and centrifugedat 100,000 g for 20 mins at 4° C. The pellet was resuspended in 100 μl50 mM Tris Ph 7.4

HEK 293T Cell Culture

HEK293T cells that stably express YFP labeled WT α-syn and A53T α-synwere a generous gift from Dr. Marc Diamond. Cells were grown inDulbecco's modified Eagle's medium (Gibco) supplemented with 10% fetalbovine serum (HyClone), 1% penicillin/streptomycin (Gibco), and 1%glutamax (Gibco) in a humidified incubator in 37° C., 5% CO2.

Seeding in HEK293T Cells

10,000 cells in 90 μL media were plated in 96 well black wall plate (Cat#3660) and allowed to adhere overnight. α-syn was transfected at a finalmonomer concentration of 125 nM. Lipofectamine 2000 was diluted inOptiMEM media (2.5+17.5 μL) and incubated at room temperature for 5mins. Protein aggregates were diluted in OptiMEM media (1:20) andsonicated in a water bath sonicator for 3 mins at low pulse. Dilutedlipofectamine and protein samples were then mixed 1:1 and incubated atroom temperature for 20 mins and thereafter 10 μL was added to eachwell. All samples are added in triplicates and experiments were repeateda minimum of two times. For co-transfection of α-syn fibrils withinhibitors, the fibrils and inhibitors were diluted in OptiMEM andincubated for 3 hours followed by sonication.

Measurement of Intracellular Puncta in Cells

Puncta formation and cell growth was measured using Celigo Imaging CellCytometer allowing for unbiased measurement. Wells were imaged usingfluorescent GFP channel and confluence was measured using Celigoanalysis software. Images of entire well were taken and particlescounted by ImageJ by particle analysis. Same settings were used toanalyze wells of one plate at all days. Total particles counted in eachwell were normalized against the confluence and reported as particlesper well.

Fibril Formation and Detection

Purified α-syn in 50 mM Tris, 150 mM KCl pH 7.5 was shaken at aconcentration of 500 μM at 37° C. in Torey Pine shaker. To form thefibrillar samples of SubNACore and NACore, lyophilized peptides weredissolved to a final concentration of 500 μM in 5 mM lithium hydroxide,20 mM sodium phosphate pH 7.5 and 0.1 M NaCl All samples were shaken at37° C. in a Torey Pine shaker for 72 hours. Freshly dissolved sampleswere prepared by dissolving lyophilized peptides immediately prior toaddition to cells for assays.

Turbidity measurements were used to compare NACore and SubNACoreaggregation. Peptide samples were freshly dissolved to 1.6 mM in asample buffer with 5 mM LiOH and 1% DMSO and then filtered through aPVDF filter (Millipore, 0.1 μm). Measurements were performed using ablack NUNC 96 well plate with 2004, of sample/well (3-4 replicates persample). The plate was agitated at 37° C., with a 3 mm rotationdiameter, at 300 rpm in a Varioskan microplate reader (Thermo).Absorbance readings were recorded every 3-15 minutes at 340 nm.

Negative Stain Transmission Electron Microscopy

Cytotxicity samples were evaluated for presence of fibrils by electronmicroscopy. Briefly, 5 μL samples were spotted directly on freshlyglow-discharged carbon-coated electron microscopy grids (Ted Pella,Redding, Calif.). After 4 min incubation, grids were rinsed twice with5-4, distilled water and stained with 2% uranyl acetate for 1 min.Specimens were examined on an FEI T12 electron microscope.

Fibril Diffraction

Fibrils formed from purified α-syn with and without N-terminalacetylation were concentrated by centrifugation, washed, and orientedwhile drying between two glass capillaries. Likewise, NACorenanocrystals were also concentrated, washed, and allowed to orient whiledrying between two glass capillaries. Aligned fibrils or nanocrystalswere mounted onto a cryopin for diffraction using 1.54 Å x-rays producedby a Rigaku rotating anode generator equipped with an HTC imaging plateand a built in X-Stream cryocooling device. All patterns were collectedat a distance of 180 mm and analyzed using the Adxv software package.

Cytotoxicity Assays

Adherent PC12 cells were cultured in ATCC-formulated RPMI 1640 medium(ATCC; cat. #30-2001) supplemented with 10% horse serum and 5% fetalbovine serum and plated at 10,000 per well to a final volume of 90 μl.All MTT assays were performed with Cell Titer 96 aqueous non-radioactivecell proliferation kit (MTT, Promega cat #4100). Cells were cultured in96-well plates for 20h at 37° C. in 5% CO2 prior to addition of samples(Costar cat. #3596). 10 μL of sample was added to each well containing90 μL medium and incubated for 24h at 37° C. in 5% CO2. Then, 15 μL dyesolution (Promega cat #4102) was added into each well, followed byincubation for 4h at 37° C. in 5% CO2. This was followed by the additionof 100 μl solubilization Solution/Stop Mix (Promega cat #4101) to eachwell. After 12h incubation at room temperature, the absorbance wasmeasured at 570 nm. Background absorbance was recorded at 700 nm. Thedata was normalized with cells treated with 1% (w/v) SDS to 0%reduction, and cells treated with sample buffer to 100% reduction.

Lactose dehydrogenase assays were done using CytoTox-ONE™ HomogeneousMembrane Integrity, (Promega, cat #G7890) as per manufacturersinstructions. Briefly, cells were plated in 96 well black-wall, clearbottom (Fisher Cat #07-200-588) tissue culture plates at 10,000 cellsper well to a final volume of 90 μL. Cells were incubated for anadditional 20h at 37° C. in 5% CO2 prior to addition of samples. Next,10 μl of sample was added to each well following which the cells wereincubated for another 24 hours. 100 μl of reagent was added to each welland incubated for 15 mins at room temperature. The addition of 50 μL ofstop solution stopped the reaction. Fluorescence was measured usingexcitation and emission wavelengths of 560 nm and 590 nm, respectively.Data was normalized using cells treated with buffer as 0% release and0.1% triton X-100 as 100% release.

The atomic coordinates of the structure of NACore: GAVVTGVTAVA (SEQ IDNO:46) below are in Table 3. The atomic coordinates of PreNAC below arein Table 4.

The atomic structure of NACore reveals an amyloid fibril formed from aself-complementary pair of β-sheets, tightly mating to form a stericzipper. By Rossetta-based computational modeling, each of the inhibitorswas designed to bind to the tip of the steric zipper and thus ‘cap’ thefibrils, preventing further molecules of alpha-synuclein to add to thefibrils. We designed 3 candidate inhibitors; S37, S61 and S71 that bindfavorably to one or both ends of the zipper. The computed bindingenergies and shape complementarity of each of the three inhibitors withthe fibril are also favorable for binding. Each of the inhibitorsretains most residues of the native sequence of NACore but also containone or more modified residues. Rodriquez et al. showed that a smaller9-residue segment within NACore [69-77] aggregates slower than NACoreand the structure is similar to NACore. In order to prevent theself-aggregation of our designed inhibitors, we used the shorter segmentalong with one or more modifications. S37 has a W mutation at Thr72 andan additional poly-lysine tag at the C-terminus to induce charge-chargerepulsion. It is predicted to bind both tips of the steric zipper fibril(i.e. top and bottom). S61 and S62 retain the same inhibitor sequence asS37 but instead of poly-lysine tag, a TAT tag is added to aid solubilityand prevent self-aggregation. S71 has a methylated glycine at Gly73 thatweakens hydrogen bonding along the β-sheet and an additional TAT tag forsolubility and cell penetration.

Example II: Computational Design

Based on the atomic structure of a fiber forming segment of α-synuclein,we designed a series of inhibitors that inhibit aggregation. We designedpeptidic inhibitors to specifically “cap” the growing aggregates ofα-synuclein. Using the ZipperDB algorithm (Goldschmidt et al.⁵⁵), thecrystallizable amyloid-forming segments in the NAC domain wereidentified. This domain has been reported to be necessary and sufficientfor aggregation and toxicity of α-synuclein (Bodies et al. ²⁰). Theidentified segments (α-synuclein segments comprised of residues 69-77and residues 68-78) were chemically synthesized and crystallized; theirthree-dimensional structures were determined by micro-crystallographyand micro-electron diffraction (MicroED). We then applied aRosetta-based method (Sievers et al. ³⁶) in their steps to designinhibitors that disrupt α-synuclein aggregation, using the α-synuclein69-77 structure or the α-synuclein 68-78 structure as a template.Putative inhibitors were identified and selected for experimentalcharacterization.

Example III—Second Round of Design—Rational, Sequence-Based Design

In another round of design, we started from several alpha-synucleinamyloidogenic segments: 47-56, 68-78 and 84-94. N-methylation wasintroduced at two separate residues in the native amino acid sequence.In these N-methyl amino acids, an additional methyl group replaces theamine hydrogen. Thus it blocks the hydrogen bond and prevents theaddition of peptide monomers on its growing end. In addition toN-methylation, additional polar/charged amino acid extensions fromnative alpha-synuclein sequence were considered to improve the overallsolubility of the peptide inhibitor.

TABLE 3 TITLE ELECTRON DIFFRACTION STRUCTURE OF THE PARKINSON'S DISEASETOXIC CORE TITLE 2 OF ALPHA-SYNUCLEIN AMYLOID, GAVVTGVTAVA COMPNDMOL_ID: 1; COMPND 2 MOLECULE: ALPHA-SYNUCLEIN; COMPND 3 CHAIN: A; COMPND4 SYNONYM: NON-A BETA COMPONENT OF AD AMYLOID, NON-A4 COMPONENT OFCOMPND 5 AMYLOID PRECURSOR, NACP; COMPND 6 ENGINEERED: YES SOURCEMOL_ID: 1; SOURCE 2 SYNTHETIC: YES; SOURCE 3 ORGANISM_SCIENTIFIC: HOMOSAPIENS; SOURCE 4 ORGANISM_COMMON: HUMAN; SOURCE 5 ORGANISM_TAXID: 9606;SOURCE 6 OTHER_DETAILS: SYNTHETIC PEPTIDE GAVVTGVTAVA CORRESPONDING TOSOURCE 7 SEGMENT 68-78 OF HUMAN ALPHA-SYNUCLEIN KEYWDS AMYLOID,ALPHA-SYNUCLEIN, PARKINSON'S DISEASE, TOXIC CORE, NACORE, KEYWDS 2 LIPIDBINDING PROTEIN EXPDTA ELECTRON CRYSTALLOGRAPHY AUTHOR J. A. RODRIGUEZ,M. IVANOVA, M. R. SAWAYA, D. CASCIO, F. REYES, D. SHI, L. JOHNSON,AUTHOR 2 E. GUENTHER, S. SANGWAN, J. HATTNE, B. NANNENGA, A. S.BREWSTER, AUTHOR 3 M. MESSERSCHMIDT, S. BOUTET, N. K. SAUTER, T. GONEN,D. S. EISENBERG JRNL AUTH J. A. RODRIGUEZ, M. IVANOVA, M. R. SAWAYA, D.CASCIO, F. REYES, D. SHI, JRNL AUTH 2 L. JOHNSON, E. GUENTHER, S.SANGWAN, J. HATTNE, B. NANNENGA, JRNL AUTH 3 A. S. BREWSTER, M.MESSERSCHMIDT, S. BOUTET, N. K. SAUTER, T. GONEN, JRNL AUTH 4 D. S.EISENBERG JRNL TITL MICROED STRUCTURE OF THE TOXIC CORE OFALPHA-SYNUCLEIN JRNL TITL 2 AMYLOID, THE PROTEIN ASSOCIATED WITH THEDEVELOPMENT OF JRNL TITL 3 PARKINSONS DISEASE JRNL REF TO BE PUBLISHEDJRNL REFN REMARK 2 REMARK 2 RESOLUTION. 1.43 ANGSTROMS. REMARK 3 REMARK3 REFINEMENT. REMARK 3 PROGRAM: BUSTER REMARK 3 AUTHORS: BRICOGNE,BLANC, BRANDL, FLENSBURG, KELLER, REMARK 3: PACIOREK, ROVERSI, SMART,VONRHEIN, WOMACK, REMARK 3: MATTHEWS, TEN EYCK, TRONRUD REMARK 3 REMARK3 DATA USED IN REFINEMENT. REMARK 3 RESOLUTION RANGE HIGH (ANGSTROMS):1.43 REMARK 3 RESOLUTION RANGE LOW (ANGSTROMS): 16.43 REMARK 3 DATACUTOFF (SIGMA (F)): 0.000 REMARK 3 COMPLETENESS FOR RANGE (%): 87.9REMARK 3 NUMBER OF REFLECTIONS: 1073 REMARK 3 REMARK 3 FIT TO DATA USEDIN REFINEMENT. REMARK 3 CROSS-VALIDATION METHOD: THROUGHOUT REMARK 3FREE R VALUE TEST SET SELECTION: RANDOM REMARK 3 R VALUE (WORKING + TESTSET): 0.251 REMARK 3 R VALUE (WORKING SET): 0.248 REMARK 3 FREE R VALUE:0.275 REMARK 3 FREE R VALUE TEST SET SIZE (%): 11.840 REMARK 3 FREE RVALUE TEST SET COUNT: 127 REMARK 3 ESTIMATED ERROR OF FREE R VALUE: NULLREMARK 3 REMARK 3 FIT IN THE HIGHEST RESOLUTION BIN. REMARK 3 TOTALNUMBER OF BINS USED: 5 REMARK 3 BIN RESOLUTION RANGE HIGH (ANGSTROMS):1.43 REMARK 3 BIN RESOLUTION RANGE LOW (ANGSTROMS): 1.60 REMARK 3 BINCOMPLETENESS (WORKING + TEST ) (%): 87.88 REMARK 3 REFLECTIONS IN BIN(WORKING + TEST SET): 286 REMARK 3 BIN R VALUE (WORKING + TEST SET):0.2642 1 REMARK 3 REFLECTIONS IN BIN (WORKING SET): 245 REMARK 3 BIN RVALUE (WORKING SET): 0.2532 REMARK 3 BIN FREE R VALUE: 0.3310 REMARK 3BIN FREE R VALUE TEST SET SIZE (%): 14.34 REMARK 3 BIN FREE R VALUE TESTSET COUNT: 41 REMARK 3 ESTIMATED ERROR OF BIN FREE R VALUE: NULL REMARK3 REMARK 3 NUMBER OF NON-HYDROGEN ATOMS USED IN REFINEMENT. REMARK 3PROTEIN ATOMS: 66 REMARK 3 NUCLEIC ACID ATOMS: 0 REMARK 3 HETEROGENATOMS: 0 REMARK 3 SOLVENT ATOMS: 2 REMARK 3 REMARK 3 B VALUES. REMARK 3FROM WILSON PLOT (A**2): 10.33 REMARK 3 MEAN B VALUE (OVERALL, A**2):12.75 REMARK 3 OVERALL ANISOTROPIC B VALUE. REMARK 3 B11 (A**2):−3.98760 REMARK 3 B22 (A**2): 3.49080 REMARK 3 B33 (A**2): 0.49680REMARK 3 B12 (A**2): 0.00000 REMARK 3 B13 (A**2): −1.55090 REMARK 3 B23(A**2): 0.00000 REMARK 3 REMARK 3 ESTIMATED COORDINATE ERROR. REMARK 3ESD FROM LUZZATI PLOT (A): 0.305 REMARK 3 DPI (BLOW EQ-10) BASED ON RVALUE (A): NULL REMARK 3 DPI (BLOW EQ-9) BASED ON FREE R VALUE (A): NULLREMARK 3 DPI ( CRUICKSHANK) BASED ON R VALUE (A): 0.108 REMARK 3 DPI(CRUICKSHANK) BASED ON FREE R VALUE (A): NULL REMARK 3 REMARK 3REFERENCES: BLOW, D. (2002) ACTA CRYST D58, 792-797 REMARK 3CRUICKSHANK, D. W. J. (1999) ACTA CRYST D55, 583-601 REMARK 3 REMARK 3CORRELATION COEFFICIENTS. REMARK 3 CORRELATION COEFFICIENT FO-FC: 0.913REMARK 3 CORRELATION COEFFICIENT FO-FC FREE: 0.900 REMARK 3 REMARK 3NUMBER OF GEOMETRIC FUNCTION TERMS DEFINED: 15 REMARK 3 TERM COUNTWEIGHT FUNCTION. REMARK 3 BOND LENGTHS: 65; 2.000; HARMONIC REMARK 3BOND ANGLES: 90; 2.000; HARMONIC REMARK 3 TORSION ANGLES: 16; 2.000;SINUSOIDAL REMARK 3 TRIGONAL CARBON PLANES: 1; 2.000; HARMONIC REMARK 3GENERAL PLANES: 10; 5.000; HARMONIC REMARK 3 ISOTROPIC THERMAL FACTORS:65; 20.000; HARMONIC REMARK 3 BAD NON-BONDED CONTACTS: NULL; NULL; NULLREMARK 3 IMPROPER TORSIONS: NULL; NULL; NULL REMARK 3 PSEUDOROTATIONANGLES: NULL; NULL; NULL REMARK 3 CHIRAL IMPROPER TORSION: 11; 5.000;SEMIHARMONIC REMARK 3 SUM OF OCCUPANCIES: NULL; NULL; NULL REMARK 3UTILITY DISTANCES: NULL; NULL; NULL REMARK 3 UTILITY ANGLES: NULL; NULL;NULL REMARK 3 UTILITY TORSION: NULL; NULL; NULL REMARK 3 IDEAL-DISTCONTACT TERM: 76; 4.000; SEMIHARMONIC REMARK 3 REMARK 3 RMS DEVIATIONSFROM IDEAL VALUES. REMARK 3 BOND LENGTHS (A): 0.010 REMARK 3 BOND ANGLES(DEGREES): 1.65 REMARK 3 PEPTIDE OMEGA TORSION ANGLES (DEGREES): 4.08REMARK 3 OTHER TORSION ANGLES (DEGREES): 6.49 REMARK 3 REMARK 3 TLSDETAILS REMARK 3 NUMBER OF TLS GROUPS: NULL 2 REMARK 3 REMARK 3 OTHERREFINEMENT REMARKS: NULL REMARK 4 REMARK 4 4RIL COMPLIES WITH FORMAT V.3.30, 13-JUL-11 REMARK 100 REMARK 100 THIS ENTRY HAS BEEN PROCESSED BYRCSB ON 09-OCT-14. REMARK 100 THE RCSB ID CODE IS RCSB087390. REMARK 240REMARK 240 EXPERIMENTAL DETAILS REMARK 240 RECONSTRUCTION METHOD: NULLREMARK 240 SAMPLE TYPE: NULL REMARK 240 SPECIMEN TYPE: NULL REMARK 240DATA ACQUISITION REMARK 240 DATE OF DATA COLLECTION: 28-AUG-14 REMARK240 TEMPERATURE (KELVIN): 100.0 REMARK 240 PH: NULL REMARK 240 NUMBER OFCRYSTALS USED: 4 REMARK 240 MICROSCOPE MODEL: TECNAI F20 TEM REMARK 240DETECTOR TYPE: TVIPS F416 CMOS CAMERA REMARK 240 ACCELERATION VOLTAGE(KV): NULL REMARK 240 NUMBER OF UNIQUE REFLECTIONS: 1073 REMARK 240RESOLUTION RANGE HIGH (A): 1.430 REMARK 240 RESOLUTION RANGE LOW (A):16.430 REMARK 240 DATA SCALING SOFTWARE: XSCALE REMARK 240 COMPLETENESSFOR RANGE (%): 89.9 REMARK 240 DATA REDUNDANCY: 4.400 REMARK 240 IN THEHIGHEST RESOLUTION SHELL REMARK 240 HIGHEST RESOLUTION SHELL, RANGE HIGH(A): 1.43 REMARK 240 HIGHEST RESOLUTION SHELL, RANGE LOW (A): 1.60REMARK 240 COMPLETENESS FOR SHELL (%): 82.5 REMARK 240 DATA REDUNDANCYIN SHELL: 4.40 REMARK 240 R MERGE FOR SHELL (I): 0.56500 REMARK 240METHOD USED TO DETERMINE THE STRUCTURE: MOLECULAR REPLACEMENT REMARK 240SOFTWARE USED: PHASER REMARK 240 STARTING MODEL: 4RIK REMARK 290 REMARK290 CRYSTALLOGRAPHIC SYMMETRY REMARK 290 SYMMETRY OPERATORS FOR SPACEGROUP: C 1 2 1 REMARK 290 REMARK 290 SYMOP SYMMETRY REMARK 290 NNNMMMOPERATOR REMARK 290 1555 X, Y, Z REMARK 290 2555 −X, Y, −Z REMARK 2903555 X + 1/2, Y + 1/2, Z REMARK 290 4555 −X + 1/2, Y + 1/2, −Z REMARK290 REMARK 290 WHERE NNN −> OPERATOR NUMBER REMARK 290 MMM −>TRANSLATION VECTOR REMARK 290 REMARK 290 CRYSTALLOGRAPHIC SYMMETRYTRANSFORMATIONS REMARK 290 THE FOLLOWING TRANSFORMATIONS OPERATE ON THEATOM/HETATM REMARK 290 RECORDS IN THIS ENTRY TO PRODUCECRYSTALLOGRAPHICALLY REMARK 290 RELATED MOLECULES. REMARK 290 SMTRY1 11.000000 0.000000 0.000000 0.00000 REMARK 290 SMTRY2 1 0.000000 1.0000000.000000 0.00000 REMARK 290 SMTRY3 1 0.000000 0.000000 1.000000 0.00000REMARK 290 SMTRY1 2 −1.000000 0.000000 0.000000 0.00000 REMARK 290SMTRY2 2 0.000000 1.000000 0.000000 0.00000 REMARK 290 SMTRY3 2 0.0000000.000000 −1.000000 0.00000 REMARK 290 SMTRY1 3 1.000000 0.0000000.000000 35.40500 REMARK 290 SMTRY2 3 0.000000 1.000000 0.000000 2.41000REMARK 290 SMTRY3 3 0.000000 0.000000 1.000000 0.00000 REMARK 290 SMTRY14 −1.000000 0.000000 0.000000 35.40500 REMARK 290 SMTRY2 4 0.0000001.000000 0.000000 2.41000 3 REMARK 290 SMTRY3 4 0.000000 0.0000001.000000 0.00000 REMARK 290 REMARK 290 REMARK: NULL REMARK 300 REMARK300 BIOMOLECULE: 1, 2 REMARK 300 SEE REMARK 350 FOR THE AUTHOR PROVIDEDAND/OR PROGRAM REMARK 300 GENERATED ASSEMBLY INFORMATION FOR THESTRUCTURE IN REMARK 300 THIS ENTRY. THE REMARK MAY ALSO PROVIDEINFORMATION ON REMARK 300 BURIED SURFACE AREA. REMARK 300 REMARK: THEBIOLOGICAL UNIT IS A PAIR OF BETA-SHEETS. ONE SHEET IS REMARK 300COMPOSED OF CHAIN A AND UNIT CELL TRANSLATIONS ALONG THE B REMARK 300DIMENSION. THE OTHER SHEET IS COMPOSED OF THE SYMMETRY MATE − X + 1/2,REMARK 300 Y + 1/2, −Z, AND UNIT CELL TRANSLATIONS ALONG B. REMARK 350REMARK 350 COORDINATES FOR A COMPLETE MULTIMER REPRESENTING THE KNOWNREMARK 350 BIOLOGICALLY SIGNIFICANT OLIGOMERIZATION STATE OF THE REMARK350 MOLECULE CAN BE GENERATED BY APPLYING BIOMT TRANSFORMATIONS REMARK350 GIVEN BELOW. BOTH NON-CRYSTALLOGRAPHIC AND REMARK 350CRYSTALLOGRAPHIC OPERATIONS ARE GIVEN. REMARK 350 REMARK 350BIOMOLECULE: 1 REMARK 350 AUTHOR DETERMINED BIOLOGICAL UNIT: TETRAMERICREMARK 350 APPLY THE FOLLOWING TO CHAINS: A REMARK 350 BIOMT1 1 1.0000000.000000 0.000000 0.00000 REMARK 350 BIOMT2 1 0.000000 1.000000 0.0000000.00000 REMARK 350 BIOMT3 1 0.000000 0.000000 1.000000 0.00000 REMARK350 BIOMT1 2 1.000000 0.000000 0.000000 0.00000 REMARK 350 BIOMT2 20.000000 1.000000 0.000000 4.82000 REMARK 350 BIOMT3 2 0.000000 0.0000001.000000 0.00000 REMARK 350 BIOMT1 3 −1.000000 0.000000 0.00000035.40500 REMARK 350 BIOMT2 3 0.000000 1.000000 0.000000 2.41000 REMARK350 BIOMT3 3 0.000000 0.000000 −1.000000 0.00000 REMARK 350 BIOMT1 4−1.000000 0.000000 0.000000 35.40500 REMARK 350 BIOMT2 4 0.0000001.000000 0.000000 7.23000 REMARK 350 BIOMT3 4 0.000000 0.000000−1.000000 0.00000 REMARK 350 REMARK 350 BIOMOLECULE: 2 REMARK 350 AUTHORDETERMINED BIOLOGICAL UNIT: MONOMERIC REMARK 350 APPLY THE FOLLOWING TOCHAINS: A REMARK 350 BIOMT1 1 1.000000 0.000000 0.000000 0.00000 REMARK350 BIOMT2 1 0.000000 1.000000 0.000000 0.00000 REMARK 350 BIOMT3 10.000000 0.000000 1.000000 0.00000 DBREF 4 RIL A 1 11 UNP P37840SYUA_HUMAN 68 78 SEQRES 1 A 11 GLY ALA VAL VAL THR GLY VAL THR ALA VALALA FORMUL 2 HOH *2 (H2 O) CRYST1 70.810 4.820 16.790 90.00 105.68 90.00C 1 2 1 4 ORIGX1 1.000000 0.000000 0.000000 0.00000 ORIGX2 0.0000001.000000 0.000000 0.00000 ORIGX3 0.000000 0.000000 1.000000 0.00000SCALE1 0.014122 0.000000 0.003964 0.00000 SCALE2 0.000000 0.2074690.000000 0.00000 SCALE3 0.000000 0.000000 0.061861 0.00000 ATOM 1 N GLYA 1 −1.664 2.302 6.349 1.00 23.58 N ATOM 2 CA GLY A 1 −1.194 2.605 5.0071.00 23.02 C ATOM 3 C GLY A 1 0.105 1.901 4.702 1.00 21.89 C ATOM 4 OGLY A 1 0.121 0.676 4.579 1.00 20.14 O ATOM 5 N ALA A 2 1.197 2.6664.558 1.00 16.28 N ATOM 6 CA ALA A 2 2.509 2.094 4.221 1.00 15.41 C ATOM7 C ALA A 2 3.664 2.731 5.005 1.00 12.05 C ATOM 8 O ALA A 2 3.668 3.9425.227 1.00 10.77 O ATOM 9 CB ALA A 2 2.764 2.186 2.712 1.00 15.67 C ATOM10 N VAL A 3 4.640 1.905 5.417 1.00 5.98 N ATOM 11 CA VAL A 3 5.8522.304 6.129 1.00 3.93 C ATOM 12 C VAL A 3 7.023 1.700 5.344 1.00 5.93 C4 ATOM 13 O VAL A 3 7.217 0.485 5.388 1.00 6.66 O ATOM 14 CB VAL A 35.856 1.891 7.629 1.00 7.66 C ATOM 15 CG1 VAL A 3 7.148 2.337 8.330 1.006.70 C ATOM 16 CG2 VAL A 3 4.661 2.494 8.333 1.00 6.73 C ATOM 17 N VAL A4 7.702 2.522 4.533 1.00 3.26 N ATOM 18 CA VAL A 4 8.782 2.087 3.6381.00 4.61 C ATOM 19 C VAL A 4 10.065 2.743 4.088 1.00 4.32 C ATOM 20 OVAL A 4 10.149 3.973 4.071 1.00 3.00 O ATOM 21 CB VAL A 4 8.458 2.4572.171 1.00 8.53 C ATOM 22 CGI VAL A 4 9.486 1.874 1.188 1.00 9.58 C ATOM23 CG2 VAL A 4 7.041 2.046 1.790 1.00 9.20 C ATOM 24 N THR A 5 11.0661.952 4.438 1.00 3.32 N ATOM 25 CA THR A 5 12.360 2.426 4.896 1.00 3.88C ATOM 26 C THR A 5 13.504 1.769 4.092 1.00 5.43 C ATOM 27 O THR A 513.567 0.539 4.039 1.00 6.20 O ATOM 28 CB THR A 5 12.540 2.051 6.3511.00 6.84 C ATOM 29 OG1 THR A 5 11.414 2.503 7.130 1.00 9.13 O ATOM 30CG2 THR A 5 13.891 2.524 6.943 1.00 10.98 C ATOM 31 N GLY A 6 14.4172.588 3.546 1.00 6.04 N ATOM 32 CA GLY A 6 15.571 2.100 2.793 1.00 6.46C ATOM 33 C GLY A 6 16.874 2.688 3.292 1.00 7.38 C ATOM 34 O GLY A 616.959 3.896 3.501 1.00 6.32 O ATOM 35 N VAL A 7 17.913 1.864 3.436 1.004.11 N ATOM 36 CA VAL A 7 19.223 2.311 3.884 1.00 5.04 C ATOM 37 C VAL A7 20.243 1.730 2.870 1.00 4.68 C ATOM 38 O VAL A 7 20.244 0.519 2.6781.00 5.69 O ATOM 39 CB VAL A 7 19.532 1.802 5.294 1.00 9.15 C ATOM 40CGI VAL A 7 21.007 1.991 5.629 1.00 9.39 C ATOM 41 CG2 VAL A 7 18.6062.431 6.367 1.00 9.75 C ATOM 42 N THR A 8 21.105 2.570 2.279 1.00 3.68 NATOM 43 CA THR A 8 22.201 2.169 1.357 1.00 4.35 C ATOM 44 C THR A 823.468 2.783 1.930 1.00 6.01 C ATOM 45 O THR A 8 23.530 4.005 2.031 1.006.48 O ATOM 46 CB THR A 8 21.874 2.496 −0.083 1.00 12.43 C ATOM 47 OG1THR A 8 20.601 1.921 −0.394 1.00 14.76 O ATOM 48 CG2 THR A 8 22.9291.954 −1.075 1.00 13.32 C ATOM 49 N ALA A 9 24.453 1.977 2.339 1.00 7.04N ATOM 50 CA ALA A 9 25.628 2.542 2.994 1.00 9.57 C ATOM 51 C ALA A 926.967 1.849 2.849 1.00 11.59 C ATOM 52 O ALA A 9 27.052 0.628 2.7561.00 8.18 O ATOM 53 CB ALA A 9 25.328 2.729 4.478 1.00 10.30 C ATOM 54 NVAL A 10 28.017 2.646 2.956 1.00 10.58 N ATOM 55 CA VAL A 10 29.4052.206 3.043 1.00 12.39 C ATOM 56 C VAL A 10 29.896 2.880 4.314 1.0018.51 C ATOM 57 O VAL A 10 30.171 4.075 4.301 1.00 16.08 O ATOM 58 CBVAL A 10 30.273 2.561 1.816 1.00 17.83 C ATOM 59 CGI VAL A 10 31.7442.243 2.085 1.00 18.89 C ATOM 60 CG2 VAL A 10 29.778 1.831 0.567 1.0017.39 C ATOM 61 N ALA A 11 29.916 2.136 5.427 1.00 17.81 N ATOM 62 CAALA A 11 30.340 2.647 6.726 1.00 23.47 C ATOM 63 C ALA A 11 31.643 1.9887.097 1.00 51.68 C ATOM 64 O ALA A 11 31.622 0.916 7.734 1.00 58.63 OATOM 65 CB ALA A 11 29.289 2.350 7.778 1.00 24.49 C ATOM 66 OXT ALA A 1132.691 2.485 6.653 1.00 74.50 O TER 67 ALA A 11 HETATM 68 O HOH A 1019.830 4.990 7.085 1.00 11.92 O HETATM 69 O HOH A 102 18.548 3.873 0.2601.00 20.36 O MASTER 203 0 0 0 0 0 0 6 68 1 0 1 END 5

The atomic coordinates of the structure of PreNAC: GVVHGVTTVA (SEQ IDNO:47) below in Table 4

TABLE 4 HEADER ---- 27-APR-15 xxxx COMPND --- REMARK 3 REMARK 3REFINEMENT. REMARK 3 PROGRAM: REFMAC 5.8.0073 REMARK 3 AUTHORS:MURSHUDOV, SKUBAK, LEBEDEV, PANNU, REMARK 3 STEINER, NICHOLLS, WINN,LONG, VAGIN REMARK 3 REMARK 3 REFINEMENT TARGET: MAXIMUM LIKELIHOODREMARK 3 REMARK 3 DATA USED IN REFINEMENT. REMARK 3 RESOLUTION RANGEHIGH (ANGSTROMS): 1.41 REMARK 3 RESOLUTION RANGE LOW (ANGSTROMS): 32.94REMARK 3 DATA CUTOFF (SIGMA (F)): NONE REMARK 3 COMPLETENESS FOR RANGE(%): 86.73 REMARK 3 NUMBER OF REFLECTIONS: 1006 REMARK 3 REMARK 3 FIT TODATA USED IN REFINEMENT. REMARK 3 CROSS-VALIDATION METHOD: THROUGHOUTREMARK 3 FREE R VALUE TEST SET SELECTION: RANDOM REMARK 3 R VALUE(WORKING + TEST SET): 0.26104 REMARK 3 R VALUE (WORKING SET): 0.25243REMARK 3 FREE R VALUE: 0.33523 REMARK 3 FREE R VALUE TEST SET SIZE (%):10.0 REMARK 3 FREE R VALUE TEST SET COUNT: 112 REMARK 3 REMARK 3 FIT INTHE HIGHEST RESOLUTION BIN. REMARK 3 TOTAL NUMBER OF BINS USED: 20REMARK 3 BIN RESOLUTION RANGE HIGH: 1.409 REMARK 3 BIN RESOLUTION RANGELOW: 1.446 REMARK 3 REFLECTION IN BIN (WORKING SET): 49 REMARK 3 BINCOMPLETENESS (WORKING + TEST ) (%): 51.43 REMARK 3 BIN R VALUE (WORKINGSET): 0.388 REMARK 3 BIN FREE R VALUE SET COUNT: 5 REMARK 3 BIN FREE RVALUE: 0.528 REMARK 3 REMARK 3 NUMBER OF NON-HYDROGEN ATOMS USED INREFINEMENT. REMARK 3 ALL ATOMS: 68 REMARK 3 REMARK 3 B VALUES. REMARK 3FROM WILSON PLOT (A**2): NULL REMARK 3 MEAN B VALUE (OVERALL, A**2):19.367 REMARK 3 OVERALL ANISOTROPIC B VALUE. REMARK 3 B11 (A**2): −0.23REMARK 3 B22 (A**2): −0.23 REMARK 3 B33 (A**2): 0.49 REMARK 3 B12(A**2): 0.00 REMARK 3 B13 (A**2): −0.30 REMARK 3 B23 (A**2): −0.00REMARK 3 REMARK 3 ESTIMATED OVERALL COORDINATE ERROR. REMARK 3 ESU BASEDON R VALUE (A): 0.118 REMARK 3 ESU BASED ON FREE R VALUE (A): 0.133REMARK 3 ESU BASED ON MAXIMUM LIKELIHOOD (A): 0.155 REMARK 3 ESU FOR BVALUES BASED ON MAXIMUM LIKELIHOOD (A**2): 4.934 REMARK 3 REMARK 3CORRELATION COEFFICIENTS. REMARK 3 CORRELATION COEFFICIENT FO-FC: 0.950REMARK 3 CORRELATION COEFFICIENT FO-FC FREE: 0.895 REMARK 3 REMARK 3 RMSDEVIATIONS FROM IDEAL VALUES COUNT RMS WEIGHT REMARK 3 BOND LENGTHSREFINED ATOMS (A): 66; 0.017; 0.019 REMARK 3 BOND LENGTHS OTHERS (A):69; 0.010; 0.020 REMARK 3 BOND ANGLES REFINED ATOMS (DEGREES): 91;1.876; 1.905 1 REMARK 3 BOND ANGLES OTHERS (DEGREES): 155; 0.648; 3.000REMARK 3 TORSION ANGLES, PERIOD 1 (DEGREES): 9; 5.684; 5.000 REMARK 3TORSION ANGLES, PERIOD 2 (DEGREES): 1; 75.981; 20.000 REMARK 3 TORSIONANGLES, PERIOD 3 (DEGREES): 7; 7.001; 15.000 REMARK 3 CHIRAL-CENTERRESTRAINTS (A**3): 14; 0.098; 0.200 REMARK 3 GENERAL PLANES REFINEDATOMS (A): 73; 0.005; 0.020 REMARK 3 GENERAL PLANES OTHERS (A): 13;0.001; 0.020 REMARK 3 REMARK 3 ISOTROPIC THERMAL FACTOR RESTRAINTS.COUNT RMS WEIGHT REMARK 3 MAIN-CHAIN BOND REFINED ATOMS (A**2): 39;6.545; 1.715 REMARK 3 MAIN-CHAIN BOND OTHER ATOMS (A**2): 38; 3.948;1.607 REMARK 3 MAIN-CHAIN ANGLE REFINED ATOMS (A**2): 41; 8.395; 2.554REMARK 3 MAIN-CHAIN ANGLE OTHER ATOMS (A**2): 42; 8.555; 2.605 REMARK 3SIDE-CHAIN BOND REFINED ATOMS (A**2): 27; 5.989; 2.165 REMARK 3SIDE-CHAIN BOND OTHER ATOMS (A**2): 27; 5.243; 2.077 REMARK 3 SIDE-CHAINANGLE OTHER ATOMS (A**2): 44; 8.091; 3.056 REMARK 3 LONG RANGE B REFINEDATOMS (A**2): 54; 9.624; 14.765 REMARK 3 LONG RANGE B OTHER ATOMS (A**2)55; 11.399; 15.221 REMARK 3 REMARK 3 NCS RESTRAINTS STATISTICS REMARK 3NUMBER OF NCS GROUPS: NULL REMARK 3 REMARK 3 TWIN DETAILS REMARK 3NUMBER OF TWIN DOMAINS: NULL REMARK 3 REMARK 3 REMARK 3 TLS DETAILSREMARK 3 NUMBER OF TLS GROUPS: NULL REMARK 3 REMARK 3 REMARK 3 BULKSOLVENT MODELLING. REMARK 3 METHOD USED: MASK REMARK 3 PARAMETERS FORMASK CALCULATION REMARK 3 VDW PROBE RADIUS: 1.20 REMARK 3 ION PROBERADIUS: 0.80 REMARK 3 SHRINKAGE RADIUS: 0.80 REMARK 3 REMARK 3 OTHERREFINEMENT REMARKS: REMARK 3 HYDROGENS HAVE BEEN ADDED IN THE RIDINGPOSITIONS REMARK 3 U VALUES: REFINED INDIVIDUALLY REMARK 3 CRYST1 17.9304.710 33.030 90.00 94.33 90.00 P 1 21 1 SCALE1 0.055772 0.0000000.004219 0.00000 SCALE2 −0.000000 0.212314 0.000000 0.00000 SCALE30.000000 −0.000000 0.030362 0.00000 ATOM 1 N GLY A 47 −7.911 −0.63918.278 1.00 20.19 N ATOM 2 CA GLY A 47 −6.901 0.239 18.839 1.00 16.21 CATOM 3 C GLY A 47 −5.632 −0.528 19.111 1.00 16.40 C ATOM 4 O GLY A 47−5.600 −1.727 19.011 1.00 22.51 O ATOM 5 N VAL A 48 −4.568 0.169 19.4441.00 17.59 N ATOM 6 CA VAL A 48 −3.313 −0.460 19.729 1.00 15.77 C ATOM 7CB VAL A 48 −2.269 −0.088 18.637 1.00 17.32 C ATOM 8 CGI VAL A 48 −0.855−0.462 19.056 1.00 17.64 C ATOM 9 CG2 VAL A 48 −2.659 −0.731 17.326 1.0018.74 C ATOM 10 C VAL A 48 −2.906 0.080 21.062 1.00 13.82 C ATOM 11 OVAL A 48 −3.040 1.262 21.327 1.00 18.65 O ATOM 12 N VAL A 49 −2.365−0.782 21.895 1.00 16.57 N ATOM 13 CA VAL A 49 −1.779 −0.342 23.136 1.0016.11 C ATOM 14 CB VAL A 49 −2.592 −0.887 24.317 1.00 16.48 C ATOM 15CGI VAL A 49 −1.861 −0.622 25.628 1.00 20.39 C ATOM 16 CG2 VAL A 49−4.002 −0.324 24.303 1.00 20.73 C ATOM 17 C VAL A 49 −0.366 −0.88223.250 1.00 13.58 C ATOM 18 O VAL A 49 −0.178 −2.043 23.137 1.00 20.25 OATOM 19 N HIS A 50 0.614 −0.063 23.557 1.00 16.22 N 2 ATOM 20 CA HIS A50 2.002 −0.524 23.593 1.00 15.91 C ATOM 21 CB HIS A 50 2.612 −0.17622.220 1.00 18.19 C ATOM 22 CG HIS A 50 4.036 −0.543 22.024 1.00 23.24 CATOM 23 ND1 HIS A 50 4.728 −1.390 22.851 1.00 32.41 N ATOM 24 CE1 HIS A50 5.964 −1.539 22.399 1.00 29.90 C ATOM 25 NE2 HIS A 50 6.088 −0.83921.291 1.00 34.03 N ATOM 26 CD2 HIS A 50 4.892 −0.215 21.028 1.00 33.27C ATOM 27 C HIS A 50 2.680 0.171 24.736 1.00 14.51 C ATOM 28 O HIS A 502.686 1.396 24.776 1.00 21.97 O ATOM 29 N GLY A 51 3.213 −0.580 25.6891.00 15.69 N ATOM 30 CA GLY A 51 3.877 0.021 26.829 1.00 11.67 C ATOM 31C GLY A 51 2.957 0.800 27.740 1.00 12.93 C ATOM 32 O GLY A 51 2.9672.022 27.786 1.00 20.51 O ATOM 33 N VAL A 52 2.174 0.081 28.498 1.0015.10 N ATOM 34 CA VAL A 52 1.315 0.692 29.514 1.00 14.31 C ATOM 35 CBVAL A 52 −0.162 0.399 29.278 1.00 14.02 C ATOM 36 CG1 VAL A 52 −1.0020.803 30.489 1.00 16.11 C ATOM 37 CG2 VAL A 52 −0.626 1.146 28.077 1.0015.58 C ATOM 38 C VAL A 52 1.778 0.094 30.802 1.00 13.92 C ATOM 39 O VALA 52 1.742 −1.133 30.969 1.00 20.14 O ATOM 40 N THR A 53 2.253 0.95431.698 1.00 17.96 N ATOM 41 CA THR A 53 2.886 0.481 32.943 1.00 18.30 CATOM 42 CB THR A 53 4.381 0.885 33.010 1.00 20.92 C ATOM 43 OG1 THR A 535.064 0.409 31.840 1.00 29.94 O ATOM 44 CG2 THR A 53 5.063 0.255 34.1751.00 22.40 C ATOM 45 C THR A 53 2.116 1.037 34.132 1.00 14.85 C ATOM 46O THR A 53 1.778 2.193 34.103 1.00 21.57 O ATOM 47 N THR A 54 1.8400.202 35.151 1.00 15.66 N ATOM 48 CA THR A 54 1.239 0.607 36.427 1.0014.08 C ATOM 49 CB THR A 54 −0.184 0.059 36.602 1.00 18.69 C ATOM 50 OG1THR A 54 −1.031 0.491 35.534 1.00 35.75 O ATOM 51 CG2 THR A 54 −0.7680.525 37.872 1.00 23.36 C ATOM 52 C THR A 54 2.061 −0.047 37.500 1.0012.83 C ATOM 53 O THR A 54 2.023 −1.258 37.626 1.00 22.40 O ATOM 54 NVAL A 55 2.821 0.725 38.262 1.00 14.38 N ATOM 55 CA VAL A 55 3.639 0.20339.339 1.00 13.58 C ATOM 56 CB VAL A 55 5.099 0.641 39.138 1.00 15.65 CATOM 57 CG1 VAL A 55 5.915 0.347 40.358 1.00 16.16 C ATOM 58 CG2 VAL A55 5.696 −0.078 37.939 1.00 17.66 C ATOM 59 C VAL A 55 3.100 0.75640.673 1.00 14.57 C ATOM 60 O VAL A 55 2.916 1.968 40.780 1.00 16.23 OATOM 61 N ALA A 56 2.809 −0.130 41.639 1.00 17.51 N ATOM 62 CA ALA A 562.466 0.268 43.022 1.00 26.29 C ATOM 63 CB ALA A 56 1.113 −0.279 43.4651.00 21.51 C ATOM 64 C ALA A 56 3.547 −0.222 43.971 1.00 38.92 C ATOM 65O ALA A 56 3.757 −1.425 44.111 1.00 69.25 O ATOM 66 OXT ALA A 56 4.2490.549 44.634 1.00 54.03 O HETATM 67 O HOH B 1 4.715 2.482 29.991 1.0027.67 O HETATM 68 O HOH B 2 −1.034 3.065 33.500 1.00 32.66 O 3

REFERENCES

-   1. Biere, A. L. et al. Parkinson's disease-associated    alpha-synuclein is more fibrillogenic than beta- and gamma-synuclein    and cannot cross-seed its homologs. J. Biol. Chem. 275, 34574-34579    (2000).-   2. Giasson, B. I., Murray, I. V. J., Trojanowski, J. Q. & Lee, V.    M.-Y. A Hydrophobic Stretch of 12 Amino Acid Residues in the Middle    of α-Synuclein Is Essential for Filament Assembly. J. Biol. Chem.    276, 2380-2386 (2001).-   3. Du, H.-N. et al. A peptide motif consisting of glycine, alanine,    and valine is required for the fibrillization and cytotoxicity of    human alpha-synuclein. Biochemistry (Mosc.) 42, 8870-8878 (2003).-   4. Periquet, M., Fulga, T., Myllykangas, L., Schlossmacher, M. G. &    Feany, M. B. Aggregated α-Synuclein Mediates Dopaminergic    Neurotoxicity In Vivo. J. Neurosci. 27, 3338-3346 (2007).-   5. Nannenga, B. L., Shi, D., Leslie, A. G. W. & Gonen, T.    High-resolution structure determination by continuous-rotation data    collection in MicroED. Nat. Methods 11, 927-930 (2014).-   6. Shi, D., Nannenga, B. L., Iadanza, M. G. & Gonen, T.    Three-dimensional electron crystallography of protein microcrystals.    eLife 2, e01345 (2013).-   7. Spillantini, M. G. et al. Alpha-synuclein in Lewy bodies. Nature    388, 839-840 (1997).-   8. Goedert, M., Spillantini, M. G., Del Tredici, K. & Braak, H. 100    years of Lewy pathology. Nat. Rev. Neurol. 9, 13-24 (2013).-   9. Polymeropoulos, M. H. et al. Mutation in the alpha-synuclein gene    identified in families with Parkinson's disease. Science 276,    2045-2047 (1997).-   10. Krüger, R. et al. Ala30Pro mutation in the gene encoding    alpha-synuclein in Parkinson's disease. Nat. Genet. 18, 106-108    (1998).-   11. Zarranz, J. J. et al. The new mutation, E46K, of alpha-synuclein    causes Parkinson and Lewy body dementia. Ann. Neurol. 55, 164-173    (2004).-   12. Ibáñez, P. et al. Causal relation between alpha-synuclein gene    duplication and familial Parkinson's disease. Lancet 364, 1169-1171    (2004).-   13. Singleton, A. B. et al. alpha-Synuclein locus triplication    causes Parkinson's disease. Science 302,841 (2003).-   14. Uéda, K. et al. Molecular cloning of cDNA encoding an    unrecognized component of amyloid in Alzheimer disease. Proc. Natl.    Acad. Sci. U.S.A 90, 11282-11286 (1993).-   15. Han, H., Weinreb, P. H. & Lansbury, P. T. The core Alzheimer's    peptide NAC forms amyloid fibrils which seed and are seeded by    beta-amyloid: is NAC a common trigger or target in neurodegenerative    disease? Chem. Biol. 2, 163-169 (1995).-   16. El-Agnaf, O. M. et al. Aggregates from mutant and wild-type    alpha-synuclein proteins and NAC peptide induce apoptotic cell death    in human neuroblastoma cells by formation of beta-sheet and    amyloid-like filaments. FEBS Lett. 440, 71-75 (1998).-   17. Conway, K. A., Harper, J. D. & Lansbury, P. T. Accelerated in    vitro fibril formation by a mutant alpha-synuclein linked to    early-onset Parkinson disease. Nat. Med. 4, 1318-1320 (1998).-   18. Nelson, R. et al. Structure of the cross-beta spine of    amyloid-like fibrils. Nature 435, 773-778 (2005).-   19. Sawaya, M. R. et al. Atomic structures of amyloid cross-beta    spines reveal varied steric zippers. Nature 447, 453-457 (2007).-   20. Bodies, A. M., Guthrie, D. J., Greer, B. & Irvine, G. B.    Identification of the region of non-Abeta component (NAC) of    Alzheimer's disease amyloid responsible for its aggregation and    toxicity. J. Neurochem. 78, 384-395 (2001).-   21. Nannenga, B. L. & Gonen, T. Protein structure determination by    MicroED. Curr. Opin. Struct. Biol. 27C, 24-31 (2014).-   22. Nannenga, B. L. eLife (2014). Structure of catalase determined    by MicroED. 2014; 3:e03600-   23. Yonekura, K., Kato, K., Ogasawara, M., Tomita, M. &    Toyoshima, C. Electron crystallography of ultrathin 3D protein    crystals: atomic model with charges. Proc. Natl. Acad. Sci. U.S.A    112, 3368-3373 (2015).-   24. Doyle, P. A. & Turner, P. S. Relativistic Hartree-Fock X-ray and    electron scattering factors. Acta Crystallogr. Sect. A 24, 390-397    (1968).-   25. Der-Sarkissian, A., Jao, C. C., Chen, J. & Langen, R. Structural    organization of alpha-synuclein fibrils studied by site-directed    spin labeling. J. Biol. Chem. 278, 37530-37535 (2003).-   26. Chen, M., Margittai, M., Chen, J. & Langen, R. Investigation of    alpha-synuclein fibril structure by site-directed spin labeling. J.    Biol. Chem. 282, 24970-24979 (2007).-   27. Sarafian, T. A. et al. Impairment of mitochondria in adult mouse    brain overexpressing predominantly full-length, N-terminally    acetylated human α-synuclein. PloS One 8, e63557 (2013).-   28. Caughey, B. & Lansbury, P. T. Protofibrils, pores, fibrils, and    neurodegeneration: separating the responsible protein aggregates    from the innocent bystanders. Annu. Rev. Neurosci. 26, 267-298    (2003).-   29. Danzer, K. M., Schnack, C., Sutcliffe, A., Hengerer, B. &    Gillardon, F. Functional protein kinase arrays reveal inhibition of    p-21-activated kinase 4 by alpha-synuclein oligomers. J. Neurochem.    103, 2401-2407 (2007).-   30. Karpinar, D. P. et al. Pre-fibrillar alpha-synuclein variants    with impaired beta-structure increase neurotoxicity in Parkinson's    disease models. EMBO J. 28, 3256-3268 (2009).-   31. Winner, B. et al. In vivo demonstration that alpha-synuclein    oligomers are toxic. Proc. Natl. Acad. Sci. U.S.A 108, 4194-4199    (2011).-   32. Chen, S. W. et al. Structural characterization of toxic    oligomers that are kinetically trapped during α-synuclein fibril    formation. Proc. Natl. Acad. Sci. U.S.A 112, E1994-2003 (2015).-   33. Bousset, L. et al. Structural and functional characterization of    two alpha-synuclein strains. Nat. Commun. 4, 2575 (2013).-   34. Auluck, P. K., Caraveo, G. & Lindquist, S. α-Synuclein: membrane    interactions and toxicity in Parkinson's disease. Annu. Rev. Cell    Dev. Biol. 26, 211-233 (2010).-   35. Lee, J. C., Langen, R., Hummel, P. A., Gray, H. B. &    Winkler, J. R. Alpha-synuclein structures from fluorescence    energy-transfer kinetics: implications for the role of the protein    in Parkinson's disease. Proc. Natl. Acad. Sci. U.S.A 101, 16466-36.    Sievers, S. A. et al. Structure-based design of non-natural    amino-acid inhibitors of amyloid fibril formation. Nature 475,    96-100 (2011).-   37. Otwinowski, Z. & Minor, W. in Methods in Enzymology (ed.    Charles W. Carter, J.) Volume 276, 307-326 (Academic Press, 1997).-   38. Weierstall, U., Spence, J. C. H. & Doak, R. B. Injector for    scattering measurements on fully solvated biospecies. Rev. Sci.    Instrum. 83, 035108 (2012).-   39. Hattne, J. et al. Accurate macromolecular structures using    minimal measurements from X-ray free-electron lasers. Nat. Methods    11, 545-548 (2014).-   40. Sauter, N. K., Hattne, J., Grosse-Kunstleve, R. W. & Echols, N.    New Python-based methods for data processing. Acta Crystallogr. D    Biol. Crystallogr. 69, 1274-1282 (2013).-   41. Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66,    125-132 (2010).-   42. McCoy, A. J. et al. Phaser crystallographic software. J. Appl.    Crystallogr. 40, 658-674 (2007).-   43. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of    macromolecular structures by the maximum-likelihood method. Acta    Crystallogr. D Biol. Crystallogr. 53, 240-255 (1997).-   44. Afonine, P. V. et al. Towards automated crystallographic    structure refinement with phenix.refine. Acta Crystallogr. D Biol.    Crystallogr. 68, 352-367 (2012).-   45. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and    development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66,    486-501 (2010).-   46. Delano, W. The PyMOL Molecular Graphics System. (Schrödinger    LLC). at <http://www.pymol.org>47. Jakes, R., Spillantini, M. G. &    Goedert, M. Identification of two distinct synucleins from human    brain. FEBS Lett. 345, 27-32 (1994).-   48. Johnson, M., Coulton, A. T., Geeves, M. A. & Mulvihill, D. P.    Targeted amino-terminal acetylation of recombinant proteins in E.    coli. PloS One 5, e15801 (2010).-   49. Whitelegge, J. P., Zhang, H., Aguilera, R., Taylor, R. M. &    Cramer, W. A. Full subunit coverage liquid chromatography    electrospray ionization mass spectrometry (LCMS+) of an oligomeric    membrane protein: cytochrome b(6)f complex from spinach and the    cyanobacterium Mastigocladus laminosus. Mol. Cell. Proteomics MCP 1,    816-827 (2002).-   50. Rao, J. N., Jao, C. C., Hegde, B. G., Langen, R. & Ulmer, T. S.    A combinatorial NMR and EPR approach for evaluating the structural    ensemble of partially folded proteins. J. Am. Chem. Soc. 132,    8657-8668 (2010).-   51. Brunger, A. T. Version 1.2 of the Crystallography and NMR    system. Nat. Protoc. 2, 2728-2733 (2007).-   52. Fabiola, F., Bertram, R., Korostelev, A. & Chapman, M. S. An    improved hydrogen bond potential: impact on medium resolution    protein structures. Protein Sci. Publ. Protein Soc. 11, 1415-1423    (2002).-   53. Comellas, G. et al. Structured regions of α-synuclein fibrils    include the early-onset Parkinson's disease mutation sites. J. Mol.    Biol. 411, 881-895 (2011).-   54. Vilar, M. et al. The fold of alpha-synuclein fibrils. Proc.    Natl. Acad. Sci. U.S.A. 105, 8637-8642 (2008).-   55. Goldschmidt, L., Teng, P. K., Riek, R. & Eisenberg, D.    Identifying the amylome, proteins capable of forming amyloid-like    fibrils. Proc. Natl. Acad. Sci. 107, 3487-3492 (2010).-   56. Read, R. J. Improved Fourier coefficients for maps using phases    from partial structures with errors. Acta Crystallogr. A 42, 140-149    (1986).-   57. Eisenberg, D. S. et al. Structure-based design of peptide    inhibitors of amyloid fibrillation, U.S. Pat. No. 8,754,034.-   58. Eisenberg, D. S. et al. Structure-based peptide inhibitors of    p53 aggregation as a new approach to cancer therapeutics; WO    2014/182961 (2014); US2014/037387.-   59. The CCP4 suite: programs for protein crystallography. Acta    Crystallogr D Biol-   Crystallogr 50, 760-3 (1994).-   60. Lawrence, M. C. & Colman, P. M. Shape complementarity at    protein/protein interfaces. J Mol Biol 234, 946-50 (1993).-   61. Kyte J, Doolittle RF (May 1983). “A simple method for displaying    the hydropathic character of a protein”. J. Mol. Biol. 157 (1):    105-32.-   62. Warren L. DeLano “The PyMOL Molecular Graphics System.” DeLano    Scientific LLC, San Clos, Calif., USA. http://www.pymol.org-   63. Jiang, L. et al. (2013). Structure-based discovery of    fiber-binding compounds that reduce the cytotoxicity of amyloid    beta. ELife 2013; 2e00857

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make changes andmodifications of the invention to adapt it to various usage andconditions and to utilize the present invention to its fullest extent.The preceding embodiments are to be construed as merely illustrative,and not limiting of the scope of the invention in any way whatsoever.The entire disclosure of all applications, patents, and publicationscited herein and in the figures are hereby incorporated in theirentirety by reference, particularly with regard to the information forwhich they are cited.

1. A composition of matter comprising at least one inhibitory peptidethat inhibits α-synuclein (SEQ ID NO: 1) aggregation by binding toresidues 68-78 of α-synuclein; wherein: (a) the inhibitory peptidecomprises the sequence: GAVVWGVTAVKK (SEQ ID NO: 3); or RAVVTGVTAVAE(SEQ ID NO: 4); and at least one of the amino acids in the inhibitorypeptide comprises a non-naturally occurring amino acid; and/or theinhibitory peptide is coupled to a heterologous peptide tag.
 2. Thecomposition of claim 1, wherein the inhibitory peptide comprises thesequence: (SEQ ID NO: 5) GAVVWGVTAVKKKKK; (SEQ ID NO: 6)GAVVWGVTAVKKGRKKRRQRRRPQ; or (SEQ ID NO: 7) YGRKKRRQRRRAVVTGVTAVAE.


3. The composition of claim 1, wherein the non-naturally occurring aminoacid comprises at least one of: a D-amino acid; or an amino acidcomprising a N-methyl group moiety.
 4. The composition of claim 1,wherein the heterologous peptide tag comprises: an amino acid sequencethat increases peptide solubility; an amino acid sequence thatfacilitates monitoring of the peptide; and/or an amino acid sequencethat facilitates peptide entry into a mammalian cell.
 5. The compositionof claim 4, wherein the heterologous peptide tag comprises: a pluralityof arginine residues; a plurality of lysine amino acids; and/or an aminoacid sequence TAT.
 6. The composition of claim 1, wherein the inhibitorypeptide is from 6 to 30 amino acids in length.
 7. The composition ofclaim 1, wherein the composition comprises a plurality of inhibitorypeptides.
 8. A pharmaceutical composition comprising a peptide of claim1, and a pharmaceutically acceptable carrier including a peptidestabilizing excipient.
 9. A complex comprising α-synuclein and a peptidecomposition of claim
 1. 10-20. (canceled)